Pyrimethamine is known to have antimalarial activities and used clinically in the therapy of toxoplasmosis and human immunodeficiency virus-associated pneumonia. In this study we aimed to test the effects of pyrimethamine on spermatogenesis in mice. For this aim, animals were given pyrimethamine as a single application and the doses were 5,10, 20, and 40 mg/kg. For the spermatogenic effects, the sperm shape abnormality, epididymal sperm counts, and testes weights were evaluated at the end of days 7, 14, 21, 28, and 35 after single pyrimethamine injection at the first day. Pyrimethamine increased the frequency of abnormal sperm shape for all studied weeks except the first week and its germ cell stage-specific effects correspond to spermatozoa, spermatids, and spermatocytes. It also decreased die epididymal sperm counts at the end of days 28 and 35, which corresponds to the spermatocyte stage of mouse spermatogenesis.