We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-alpha), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-alpha knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-alpha knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-alpha knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-alpha knockout testes. In conclusion, TNF-alpha knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.