Born on April 1, 1829, in Kitahara, Hino-juku, Musashi Province (present-day Hino-Honcho, Hino-shi, Tokyo), Genzaburō Inoue was the third son of Tozaemon Inoue, caretaker of Hachioji Sennin Doshin. Around 1847, he became a disciple of Kondō Shūsuke, the third head of the Tennen Rishin dojo. He trained diligently at the dojo established by Hikogoro Sato for Tennen Rishin-ryu practice alongside figures like Hijikata Toshizō, also studying under Kondō Isami and forging a strong friendship with him. After roughly a decade of rigorous training, Genzaburō attained the highest level of proficiency in Tennen Rishin-ryu in 1860, receiving the menkyo kaiden. His methodical progress sometimes led others to underestimate his abilities, resulting in the mistaken label of "inferior in both academics and martial arts." He received his academic education at a temple school near Kanekobashi in Hino-shuku, run by Hino Yoshitaka. In February 1862, Genzaburō joined the Rōshigumi with Kondō Isami and Hijikata Toshizō. Following the purge of Serizawa Kamo's faction in 1863, he was appointed assistant vice-commander of the Shinsengumi. During the 1864 Ikedaya Incident, he commanded a detachment of Hijikata's troop. Upon learning of Kondō's unit's infiltration, Genzaburō led his subordinates in storming the establishment, capturing eight rōnin and demonstrating notable valor. June 1865 saw Genzaburō named leader of the Rokuban-tai (6th Division) during a reorganization. By June 1867, when the Shinsengumi gained official recognition as a direct vassal of the Bakufu, he was promoted to deputy chief assistant. Genzaburō met his end during the Battle of Toba-Fushimi in January 1868. As the Shinsengumi retreated to Yodo Domain, they clashed with government forces at Yodo Senryo-matsu on January 5. During this combat, Genzaburō was struck by gunfire in the abdomen and died at the age of 39.