--- - |2- Our study provided direct evidence to highlight the role of Hippo signaling in regulating follicle development and indicated that AKT functions as an upstream regulator of Hippo pathway in primordial follicle activation. Our results help to further clarify the molecular mechanism of the maintenance of primordial follicles pool and suggest a new therapeutic strategy for prevention and treatment of female infertility. Clarifying the molecular mechanisms by which primordial follicles are initiated is crucial for the prevention and treatment of female infertility and ovarian dysfunction. The Hippo pathway has been proven to have a spatiotemporal correlation with the size of the primordial follicle pool in mice in our previous work. But the role and underlying mechanisms of the Hippo pathway in primordial follicle activation remain unclear. Here, the localization and expression of the core components were examined in primordial follicles before and after activation. And the effects of the Hippo pathway on primordial follicle activation were determined by genetically manipulating yes‐associated protein 1 (Yap1), the key transcriptional effector. Furthermore, an AKT specific inhibitor (MK2206) was added to determine the interaction between the Hippo pathway and AKT, an important signaling regulator of ovarian function. Results showed that the core components of the Hippo pathway were localized in both primordial and primary follicles and the expression levels of them changed significantly during the initiation of primordial follicles. Yap1 knockdown suppressed primordial follicle activation, while its overexpression led to the opposite trend. MK2206 downregulated the ratio of P‐MST/MST1 and upregulated the ratio of P‐YAP1/YAP1 significantly, whereas Yap1‐treatment had no influence on AKT. In addition, YAP1 upregulation partially rescued the suppression of the primordial follicle activation induced by MK2206. Our findings revealed that the Hippo‐YAP1 regulates primordial follicular activation, which is mediated by AKT signaling in mice, thus providing direct and new evidence to highlight the role of Hippo signaling in regulating ovarian follicles development. - 'Journal of Cellular Physiology, Volume 234, Issue 2, Page 1578-1587, February 2019. '