Yagyū Jūbei serves the Tokugawa shogunate in "Samurai Shodown: The Motion Picture." He investigates the dark god Ambrosia's resurgence and the treachery of Shiro Tokisada Amakusa, who manipulates the shogunate for her own ends. Partnering with Hanzo Hattori and Kyoshiro Senryo, he identifies the Six Holy Warriors as Amakusa's critical vulnerability, stating, "Amakusa's weakness...appears to be the Six Holy Warriors," viewing them as the sole hope to free the Tokugawa clan.

His background as a ronin is maintained; he abandoned life as a dojo instructor to pursue personal enlightenment, yet retains loyalty to the Tokugawa regime, consistent with his established role as a demon hunter and threat eliminator. In the film, he works covertly against Amakusa's tyranny, joining Hanzo and Kyoshiro to find the reincarnated Holy Warriors. He demonstrates strategic acumen and commitment to the rebellion by aiding Galford D. Weller with information about the growing resistance.

Physically, he wears an eyepatch and wields dual swords (katana and wakizashi), reflecting his signature "Modified Shadow Style of Yagyu" and mastery of Yagyū Shinkage-ryū swordsmanship. His personality combines battle-hardened seriousness and tactical vigilance with a care-free demeanor in peacetime, though the film emphasizes his vigilance and loyalty.

The film focuses solely on his role within the coalition against Amakusa, concluding with his participation in the successful revolt and Ambrosia's defeat. It does not explore his origins or post-film activities; his ultimate fate remains unrecorded.