The activation of dormant primordial follicles is a promising method to improve the fertility of premature ovarian insufficiency (POI) patients. Many experiments from both human and animal studies suggest that human platelet-rich plasma (hPRP) may restore ovarian function and promote follicle growth. However, the underlying mechanisms remain unclear. In the current study, our results demonstrate that hPRP significantly increased the number of growing follicles and promoted the proliferation of granulosa cells in cultured mouse ovaries. hPRP also significantly increased the protein levels of phosphorylated protein kinase B (p-Akt) and forkhead box O3a (p-FOXO3a), as well as the number of oocytes with FOXO3a nuclear export in cultured mouse ovaries. Immunofluorescence results showed that in vitro treatment with hPRP significantly increased the fluorescence intensity of p-Akt in oocytes. The inhibition of the phosphatidylinositol 3 kinase (PI3K)/Akt pathway by LY294002 blocked the hPRP-induced increase in the number of growing follicles in cultured mouse ovaries. Furthermore, hPRP injected i.p. or added to the medium significantly increased the number of growing follicles and the protein levels of p-Akt in the ovaries of newborn mice and in cultured human ovarian tissues. Taken together, our findings from mouse and human experiments indicate that hPRP promotes the activation of primordial follicles through the PI3K/Akt signaling pathway in oocytes.