Insulin-like factor 6 (INSL6), a member of the insulin-like superfamily, is predominantly expressed in male germ cells. Expression of the Insl6 is first detected in mouse testis at postnatal day 15, when the first wave of spermatogenesis progresses to pachytene spermatocytes. To elucidate the role of INSL6 in germ cell development, we generated Insl6-deficient mice. The majority of the Insl6-deficient males on a hybrid genetic background exhibited impaired fertility, whereas females were fertile. The number of mature sperm and sperm motility were drastically reduced in the epididymis. The reduced sperm count could be due to apoptotic death of a significant number of developing germ cells. Analysis of germ cell development during the juvenile life showed an arrest of the first wave of spermatogenesis in late meiotic prophase. RNA analysis revealed a significant decrease in expression of late meiotic- and postmeiotic-specific marker genes, whereas expression of early meiotic-specific genes remains unaffected in the Insl6^-/- testes. These results demonstrate that INSL6 is required for the progression of spermatogenesis.