Background: In 2016, the veterinarian team of Buin Zoo in Chile decided to try to increase the lion population. At that time, the zoo had three lions; two females and one male. The 9-year-old male had been vasectomized 5 years ago at the same institution for birth control. Considering the fact that in humans, vasectomy reversal has excellent reproductive outcomes, a team of human urologists, highly experienced in vasectomy reversal was contacted to perform the procedure. Case description: Surgery was performed on June 16, 2016 under general anesthesia, with the vasectomy site accessed through the previous scar localized in the lower groin. After opening the skin, dartos and tunica vaginalis, we were able to identify the previous vasectomy site. After liberating both vas ends and checking for permeability, a microsurgical anastomosis (magnification 25×) was performed. The surgery took 80 minutes with minimal bleeding, and no surgical complications were observed. After 2 weeks, the lion joined the lionesses and reproductive follow-up was started. Seven months after surgery, one lioness became pregnant, and 4 months later gave birth to two female lion cubs, with no incidents at the zoo. Both cubs were healthy and are still living at the zoo. Conclusion: Vasectomy reversal constitutes a valid perspective to reassume fertility in previous vasectomized lions.