Spermatogenesis in Drosophila commences with cell division of germline stem cells (GSCs) to produce male germline cells at the tip of the testis. However, molecular mechanisms inducing division of male GSCs have not been reported. Insulin-like peptides are known to play an essential role in stimulation of proliferation and growth of somatic cells, and it has recently been reported that such peptides promote cell division in female Drosophila GSCs. However, their effects on male germline cells have not been characterized. We found that inhibition of insulin production and insulin signaling mutations resulted in decreased numbers of germline cells in Drosophila testes. GSC numbers were maintained in young mutant males, with a gradual decrease in abundance of GSCs with age. Furthermore, in mutants, fewer germline cysts originated from GSCs and a lower frequency of GSC division was seen. Insulin signaling was found to promote cell cycle progression of the male GSCs at the G(2)/M phase. The cell volume of spermatocytes increases up to 25 times before initiation of meiosis in Drosophila. We examined whether insulin signaling extrinsically induces the greatest cell growth in Drosophila diploid cells and found that spermatocyte growth was affected in mutants. The results indicate that in addition to its function in somatic cells, insulin signaling plays an essential role in cell proliferation and growth during male Drosophila gametogenesis and that sperm production is regulated by hormonal control via insulin-like peptides.