Gonadotropin-inhibitory hormone (GnIH) plays a crucial role in regulating reproduction in the hypothalamus of poultry and has been intensely investigated since its discovery. This study aimed to assess the effects of GnIH on testicular development, as well as on reproduction-related hormone release and gene expression levels in roosters. The administration of exogenous GnIH resulted in a significant reduction in testis weight, testis volume and semen quality ( < 0.05). Additionally, exogenous GnIH significantly up-regulates the expression of , and down-regulates the expression of ( < 0.05). GnIH application also decreased the , () and ()gene expression levels. Meanwhile, by neutralizing the effects of endogenous GnIH through immunization, testicular development on day 150 in roosters was significantly promoted. Compared to the control condition, GnIH immunization significantly down-regulated the expression of the and genes ( < 0.05). In conclusion, we found that exogenous GnIH treatment inhibited testicular development, reduces gene expression, and suppressed reproductive performance in roosters. Conversely, GnIH immunization down-regulated and genes, activates the reproductive system, and promotes the reproductive activity and testicular development of roosters.