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Lions (Panthera leo) breed well under captivity, so contraception has been commonly conducted for population management, leading to a demand for a less invasive and reversible contraceptive approach in lions. In this study, we examined the efficacy of a commercial gonadotropin-releasing hormone vaccine as a method of suppressing reproductive activity in a sexually matured female lion. Under behavioral restraint, the vaccine was injected twice (days 0 and 109). After the initial vaccination, ovarian activity is still observed. After the second vaccination, contraceptive effect was confirmed for 246 days until restart of estrous cycles. We confirmed only a slight swelling around the injection site after the second vaccination. This study may suggest an alternative option for a contraceptive method in lions.