Set primarily at the beginning of Edo period Japan (early 17th century), with anthropomorphic animals replacing humans, it features a rabbit ronin, Miyamoto Usagi, who is partially based on the famous swordsman Miyamoto Musashi. Usagi wanders the land on a musha shugyo (warrior's pilgrimage) occasionally selling his services as a bodyguard.

Usagi Yojimbo is heavily influenced by Japanese cinema and has included references to the work of Akira Kurosawa (the title of the series is derived from Kurosawa's 1960 film Yojimbo) and to icons of popular Japanese cinema such as Lone Wolf and Cub, Zatoichi, and Godzilla. The series is also influenced somewhat by Groo the Wanderer by Sergio Aragonés (Sakai is the letterer for that series), but the overall tone of Usagi Yojimbo is typically less comedic.