Long-term non-surgical contraceptive methods for cats, especially community cats, are of global interest for cost-effective and humane reasons. This study aimed to investigate the effectiveness of a gonadotropin-releasing hormone (GnRH)-based vaccine for immunocontraception and to confirm its safety in intact female cats. Recombinant Salmonella typhimurium flagellin fljB (STF2)-GnRH protein was expressed in Escherichia coli. We divided female cats into vehicle control group (n = 4) and two experimental groups (100 μg injection group [n = 7] and 1000 μg injection group [n=7]), and immunized them twice intramuscularly (0.2 mL/cat at zero week and 4 weeks later into the other leg). Breeding trials started on day 120. All control cats (n = 4/4), 71% of the 100 μg injection group (n = 5/7), and 57% of the 1000 μg injection group (n = 4/7) became pregnant within 203 days after the introduction of male cats. The 1000 μg injection group had significantly a longer median time to conception following treatment (166 days) than the control (17 days, p < 0.05). Average litter size was significantly lower in the 1000 μg GnRH-vaccinated cats (2.8 ± 0.7) than in the control cats (4.5 ± 0.5, p < 0.05). Injection site reactions were not observed in any cat. The E. coli-expressed STF2-GnRH vaccine did not provide contraception in a sufficient proportion of the cats. However, it might be effective to suppress fertility through infertility vaccines before inducing permanent infertility through the trap-neuter-return.