letrozole is an aromatase inhibitor that stops the production of estrogen through interrupting the entrance of hormone androgen into a small amount of estrogen. Therefore, the current study was developed to estimate orally administrated Letrozole on the reproductive performance and relative abundance of Foxj1, PVRL3, and LPR2 mRNA in aged roosters. Fifty-five-week old ROSS 308 breeder roosters (n ¼ 18) were orally treated using letrozole. Primarily, the body weight of the animals was recorded, and they were randomly classified into three groups (n ¼ 6 birds/group) receiving different doses of Letrozole, including 0, 0.015, and 0.03 mg/kg body weight/day for three weeks. At the end of the trial, seminal traits, plasma, testicular hormone levels (testosterone, estradiol, and FSH), histopathological studies, in vitro fertility, and relative abundance of testis PVRL3, epidydimal Foxj1, and LPR2 mRNA were evaluated. Based on the results, the sperm quality variables were statistically higher in the 0.03 group compared to the controls. Greater histologic parameters, such as diameter of seminiferous tubules, thickness of seminiferous epithelium, categorized epididymal region, and in vitro fertility rates were estimated for the treated groups(p < 0.001). Plasma and testicular testosterone, estradiol concentrations, and plasma FSH levels were significantly influenced by letrozll treatment (p < 0.001). Relative mRNA transcript abundance increased for PVRL3 and decreased for Foxj1 and LPR2 in treated groups. Overall, aromatase inhibitors can enhance the reproductive performance of aged commercial broiler breeder roosters. However, it can impact endocytosis and ciliogenesis events via reducing estradiol.