The expression of protein gene product 9.5 (PGP9.5) in testes has been reported in domestic species, including pigs, sheep, and goats. The main objectives of this study were to: (1) investigate the expression pattern of PGP9.5 in stallion testes and (2) identify stage-dependent immunolabeling of PGP9.5 in stallion testes. Samples were categorized based on the age of the horses as follows: prepubertal (<1 year), pubertal (1–1.5 years), postpubertal (2–3 years), and adult (4–8 years). Immunohistochemistry was performed to investigate the expression pattern of PGP9.5 at different reproductive stages. For double staining, the goat anti-human DAZL and GATA4 antibody were used as markers for germ cells and Sertoli cells, respectively. The Western blot procedure was used to determine the cross-reactivity of the PGP9.5 antibody in horse testis tissues. During the prepubertal and pubertal stages, germ cells, which were located in the center of the seminiferous tubules, were positive for PGP9.5. During the postpubertal and adult stages, expression of PGP9.5 was found in the cytoplasm of germ cells adjacent to the basement membrane. PGP9.5-positive cells were immunolabeled with DAZL but not immunolabeled in Sertoli cells at all reproductive stages. Interestingly, Leydig cells were stained with PGP9.5 at all reproductive stages. In conclusion, PGP9.5 antibody can be used as a tool to identify and isolate PGP9.5-positive germ cells only after Leydig cells were separated from the seminiferous tubules.