Insulin like3 (INSL3, Relaxin like factor) is a critical regulator in testis translocation through Leucin rich G-protein coupled receptor 8 (Rxfp2,LGR8) during embryogenesis. In female, INSL3 and their receptor expressed in growing follicle and revealed their function in oogenesis. However, its role is not much evaluated. 6 weeks old C57BL/6 female mice used for measure the expression of INSL3 mRNA and their receptor expression by follicular stage. Follicle cell specific RNA were got from the theca cell and granulose cells which were isolated using LCM. To know the role of INSL 3 in theca cell, Rxpf2 were overexpressed during primary culture of theca cells, which were isolated from tertiary follicles after 12 hr PMSG injection and transduced the Rxfp2 using lenti virus. After 10 days of culture, the proliferation of theca cells was analyzed using EdU. Using Alzet osmotic pumps INSL3 was administered for 3 days into the ovary during superovulation induction. The INSL3 mRNA levels were significantly high in the theca cells of preovulator follicles after hCG injection. But granulose cells showed decreasing expression by growth of follicle. INSL3 stimulated the proliferation of theca cells in vitro which overexpressed the Rxpf2. By the administration of INSL3 into ovary caused the dramatical decrease the number of ovulated oocyte. Based on these results, we know that INSL3 stimulates the theca cell proliferation in as follicle stage specific manners, and estrogen is a modulator of this INSL3 mRNA expression. It suggests that the disturbance of the expression regulation of INSL3 and its receptor cause the unregulated theca cell proliferation and failing the rupture of grown follicle.