This study aimed to investigate the influence of the Kisspeptin (KP) injection on semen quality, testosterone concentration, reaction time, testes and accessory genital glands measurements of Ossimi rams. Fourteen healthy Ossimi rams were randomly divided into control group (CG) and KP treated group (KPG) (7 per each). Rams of the KPG were treated once weekly with KP (5 μg/kg body weight) for one month. Semen volume, concentration, mass motility, percentage of live and dead sperm and total abnormal sperm were recorded twice weekly after treatment for five weeks. Testes measurements including scrotal circumference (SC), testis breadth (TB) and length (TL) using measuring tape and caliper were recorded. Blood samples were collected, and ultrasound examination of the accessory genital glands was measured. Semen characteristics were significantly (p < 0.05) affected by KP injection. The KP increased the volume (59% higher), sperm concentration (89% higher), total sperm concentration (200% higher, and percentages of live sperm (25% higher) compared with CG. Moreover, KP injection decreased (p < 0.05) sperm abnormalities (50% lower) and decreased (p < 0.05) reaction time for the first mount (68% lower) compared to CG. Furthermore, KPG showed higher (p < 0.05) testosterone concentration (66% higher) in comparison with the CG. In addition, KPG had higher (p < 0.05) SC, TB, TL than the CG. In conclusion, injections of the KP enhanced the semen properties and the reproduction of Ossimi rams during the breeding season in subtropics.