Zatoichi does not feature any identifiable transsexual characters or themes. The character Osei cross-dresses as a geisha for plot-driven disguise, not as an expression of gender identity, and the narrative does not engage with transgender issues.

The film features female characters, but none are depicted engaging in or winning close-quarters physical combat against male opponents. The character disguised as a geisha who fights is revealed to be male.

The film features the iconic character Zatoichi, who has consistently been portrayed as male across all source materials and previous adaptations. In the 2003 film, Zatoichi is again portrayed as male, and no other established characters from the franchise or historical figures undergo a gender change.

The film Zatoichi (2003) adapts a long-standing Japanese character, Zatoichi, who has consistently been portrayed as Japanese. The 2003 film, set in Edo-period Japan, features Japanese actors in all major roles, including Takeshi Kitano as Zatoichi, aligning with the character's established race.