Spermatogenesis is a very complex process that can be affected by a number of factors which may lead to reduced fertility or infertility. Busulfan kills spermatogonia stem cells and disrupts the connections between Sertoli and spermatogonia cells at base layers. Moreover, vitamin D is very important in the reproductive system of men. In this study, the effect of vitamin D on biosulfur-induced azoospermia in mice was investigated. The results showed an increase in the quality of sperm parameters in the group treated with vitamin D.