The title is a typical Western reference, as in "The Lone Ranger rides again", and also suggests a reference to the 1940 Jack Benny comedy, Buck Benny Rides Again.
After opening credits underscored by the "William Tell Overture", the music segues into a lively instrumental of "Cheyenne" as the action begins. In a typical Old West frontier town with the self-contradictory name of Rising Gorge, bullets are firing from every window across the street into every other window across the street. A hail of bullets flies down one street until a stop signal turns red and the bullets hover in mid-air while a second hail of bullets shoot by on the perpendicular street. The light changes back and just as the first hail of bullets is about to start, a lone bullet "runs the red light" at even higher speed, holding up the first stream. After it passes, the first hail continues.
Inside the Gunshot Saloon with the slogan of 'Come in a get a slug', two men are standing at a bar. One man is about to drink a shot of whiskey; the second man takes out his gun and shoots the first man. The first man throws his glass into the air and the second man catches it. A scream is heard and Yosemite Sam enters the bar. All of the patrons are afraid of Sam, yelling his name in terror while the underscore plays stereotypical "villain music". Sam says, "Yeah, Yosemite Sam. The roughest, toughest, he-man stuffest hombre whose ever crossed the Rio Grande... And I don't mean Mahatma Gandhi! (See "Censorship" below for more information about this line.) Now all of you skunks clear out of here!" After firing his guns, all the patrons run out, followed by a real skunk who retorts, "My, weren't there a lot of skunks in here?" Sam turns around to see a man trying to sneak out. Sam fires his guns at the man, who then turns into a firing range walking dummy, making a "ding" every time he is hit, with a score board above keeping tally. After that, Sam asks, "Be there any livin' varmint who aims to try to tame me?" Spying Bugs Bunny, he asks again, "Well, be there?"
No one dares to challenge Yosemite Sam except Bugs, sporting a cowboy hat and rolling a cigarette. After a brief silence, Bugs replies ... "I aims ta ..." The two slowly walk towards each other. Bugs asides, "Just like Gary Cooper, huh?" Sam declares "this town ain't big enough for the both of [them]!" Bugs tries to rectify that by running off-screen and, to sound effects of hammers and saws, quickly constructs a background of modern skyscrapers in the town, but "it still ain't big enough!" Bugs and Sam then face off threatening each other with bigger guns. After Bugs pulls out the pea shooter by accident, he flicks it at Sam, but Sam corners him and tells him "All right now, you wise guy! Dance!" while firing at his feet. Bugs grabs a cane and straw hat from off-screen, and goes into a vaudeville soft shoe routine, then says "Take it, Sam!" The diminutive villain, although startled initially, quickly breaks into the same dance, and is tricked into dancing into an open mine shaft. Bugs expresses sympathy for Sam. "Poor little maroon. So trusting, so naive."
When Sam returns to the surface and orders him to "start walking", Bugs dares him to cross a line drawn with his foot. "OK, I'm a-steppin'!" Bugs continues this schtick all the way out of town to the edge of a cliff, where the unobservant Sam steps over the line and plummets toward the ground far below. Suddenly stricken with guilt, the speedy hare dashes down a roadway, beats Sam to the ground and lays down a mattress, telling the audience, "Ya know, sometimes me conscience kind-a bodders me ... but not this time!" as he pulls away the mattress. Sam smashes into the ground, and the already pint-sized bandit has been vertically flattened to a hat with legs, but he still comes up firing.
A horseback chase ensues to the tune of the "William Tell Overture", as the two ride on horses that are proportional to their own sizes. Bugs leads Sam into a tunnel, and again showing extraordinary construction talents, has time to don a painter's cap and build a brick wall at the other end, into which Sam smacks. After more chasing, Bugs stops the chase and points out that they are getting nowhere and are right back where they began. "Hey, wait a minute, Sam! We ain't gettin' nowheres! We're right back where we started!" Sam agrees.
The two decide to settle their differences by playing cards "just like in the Western pictures", with the loser being forced to leave town. "Gin rummy's mah game, Sam." Sam tells Bugs to "cut the cards", which he does using a meat cleaver. With a new deck, Bugs tricks Sam into playing a card that gives Bugs the win. "GIN! You lose!!" Bugs tries to get Sam to take the train out of town, but when the passenger car is revealed to be full of swimsuit-clad women headed on the Miami Special, Bugs fights with Sam to board the train.
Bugs prevails as usual. He leans out the train window, his face covered with lipstick from kisses, and hollers, "So long, Sammy, see ya in Miami!"
All of the home media releases of this short have Yosemite Sam's introductory boast end with, "...and I ain't no namby-pamby" instead of the original line "...and I don't mean Mahatma Ghandi". As of this writing, the original version with the reference to Mahatma Ghandi has never been released on home media or shown on television.
- On The WB, the beginning gun- and bullet-related gags are cut: the bullets stopping and proceeding at traffic lights, one cowboy shooting another and then drinking his beer as he drops dead (a similar scene in 1959's Wild 'n Woolly Hare would also be cut when aired on The WB), and Sam shooting at a cowboy like a wooden duck in a shooting gallery. The WB version also cut the shot of Bugs rolling a cigarette when Sam challenges the cowboys to fight him.
- In the original version, Yosemite Sam's intro line when he enters the saloon is, "Yeah, Yosemite Sam. The roughest, toughest, he-man-stuffest hombre to ever cross the Rio Grande. And I don't mean Mahatma Ghandi!" Around the time the short's distribution rights were sold to Associated Artists Productions, Ghandi was already assassinated, so the last part of the line ("And I don't mean Mahatma Ghandi") was changed to "And I ain't no namby-pamby". The "namby pamby" version is the version that airs frequently on television today and even on home media releases. Although audio tracks of the original line are known to exist, a new version where the original line is reinstated has never been made.
- Cartoon Network's Arabian/Middle Eastern channel cuts out the shot near the end of the interior of the Miami Express Train, which features beautiful women in swimsuits. Other Cartoon Network stations, including the United States, left this scene uncut.
- The 1947-48 dubbed ending card is said to come from this cartoon as the dubbed LT card came from "Haredevil Hare".
- This is the next-to-last Bugs Bunny release in the a.a.p. package, with the last such cartoon being "Haredevil Hare" - also the overall latest-released WB cartoon in the a.a.p. package.
- This is the first cartoon to use COLOR BY TECHNICOLOR instead of IN TECHNICOLOR, but shortly changed to Color By TECHNICOLOR starting with "Haredevil Hare".
- The Ken Champin Veterinary is a reference to animator Ken Champin, a long time animator in Freleng's unit.
- This is the last Yosemite Sam cartoon to be sold to the a.a.p. package.
- When the cartoon's copyright was sold to Associated Artists Productions, the line "And I don't mean Mahatma Ghandi" was changed to "And I ain't no namby-pamby" (See Censorship for details). The "And I ain't no namby-pamby" version is the popular version that has circulated on American TV, home media releases, and HBO Max streaming.
- "Bugs Bunny Rides Again" at SuperCartoons.net
- "Bugs Bunny Rides Again" at B99.TV
- "Bugs Bunny Rides Again" at Archive.org (with original 1948 version audio)