The primordial follicle assembly, activation and the subsequent development are critical processes for female reproduction. A limited number of primordial follicles are activated to enter the growing follicle pool each wave, and the primordial follicle pool progressively diminishes over a woman's life-time. The number of remaining primordial follicles represents the ovarian reserve. Identification and functional investigation of the factors involved in follicular initial recruitment will be of great significance to the understanding of the female reproduction process and ovarian ageing. In this study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the primordial follicle activation in the neonatal mouse ovary. The attenuation of SEMA6C expression by SiRNA accelerated the primordial follicle activation in the invitro ovary culture system. PI3K-AKT-rpS6 pathway was activated when SEMA6C expression was down-regulated. And the LY294002 could reverse the effect of low SEMA6C expression on primordial follicle activation. Our findings revealed that Sema6c was involved in the activation of primordial follicles, and the down-regulation of SEMA6C led to massive primordial follicle activation by interacting with the PI3K-AKT-rpS6 pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian ageing.