Sakai began his career by simply lettering comic books as he perfected his art and began working in the industry.

He also wrote and illustrated The Adventures of Nilson Groundthumper and Hermy; a comic series with a medieval setting, influenced by Sergio Aragones's Groo the Wanderer. The characters first appeared in Albedo #1 in 1984, and were subsequently featured in issues of Critters, GrimJack, Amazing Heroes and Furrlough. Stan Sakai became famous with the creation of Usagi Yojimbo, the epic saga of Miyamoto Usagi, a samurai rabbit living in late-sixteenth and early-seventeenth-century Japan.

Usagi Yojimbo, first published in 1984, continues to this day.

It progresses with Stan Sakai as the lone author and nearly sole artist (Tom Luth serves as the main colorist on the series, and Sergio Aragonés has made two small contributions to the series: the story "Broken Ritual" is based on an idea by Aragonés, and he served as a guest inker for the black-and-white version of the story "Return to Adachi Plain" that is featured in the Volume 11 trade paperback edition of Usagi Yojimbo). He also created a futuristic spinoff series Space Usagi.[10] His favorite movie is Satomi Hakkenden (1959). The Japanese American National Museum in Los Angeles's Little Tokyo presented an exhibit entitled "Year of the Rabbit: Stan Sakai's Usagi Yojimbo" from July 9 through October 30, 2011.

Sakai wrote and illustrated the story "I'm Not in Springfield Anymore!" for Bart Simpson's Treehouse of Horror #7 and illustrated the back cover of Treehouse of Horror #6. Sakai was the artist for Riblet, the back-up feature in the trade paperback of Stupid, Stupid Rat Tails.In 2013, Sakai illustrated the limited comic book series 47 Ronin, an adaptation of the famed story of the 47 Ronin written by Dark Horse Comics Publisher Mike Richardson and with Lone Wolf and Cub writer Kazuo Koikeas an editorial consultant. He resides in Pasadena, California.