Folliculogenesis is a critical process during mammalian development, but little is known about the mechanisms underlying primordial follicular formation, growth, and activation. The objective of this study was to assess the effect of continuous insulin exposure on folliculogenesis in 16.5 dpc and 3 dpp mouse ovaries in vitro. The results demonstrated that 5 microg/ml insulin promotes the phosphorylation of Akt and the primordial follicular assembly and activation process via the InsR/PI3K/Akt signaling pathway in the 16.5 dpc fetal mouse ovaries cultured in vitro. However, the presence of 5 microg/ml insulin represses the phosphorylation of Akt and the follicular assembly and activation process in the 3 dpp mouse ovaries cultured in vitro. Furthermore, in vitro cultures of 3 dpp mouse ovaries with insulin can repress the expression of Pten. In conclusion, folliculogenesis was stage-specifically regulated by insulin via the Akt signaling pathway in vitro.