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Normal development of ovarian follicles is critical for female reproduction and endocrine function. We have identified retinoic acid (RA) and the RA-degrading enzyme CYP26B1 as regulators of ovarian follicle development and showed that RA and a CYP26 inhibitor stimulated ovarian granulosa cell proliferation. The mechanism underpinning RA-dependent proliferation, however, is not known. The current study was designed to examine the role of intracellular calcium (Ca2+) signaling in mediating the effects of RA on primary mouse granulosa cell proliferation. In single-cell Ca2+imaging experiments, treatment of cultured granulosa cells with RA increased the steady-state Ca2+content of the endoplasmic reticulum (ER) stores. This correlated with increased store-operated Ca2+entry (SOCE) and enhanced inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca2+release. In proliferation assays, RA treatment or Cyp26b1knockdown stimulated proliferation, whereas Cyp26b1overexpression inhibited proliferation. When RA was given together with 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3R-dependent ER Ca2+release and SOCE, with xestospongin C, a selective IP3R- receptor antagonist, or with 3,5-bis (trifluoromethyl)pyrazole (BTP-2), a specific SOCE blocker, the stimulatory effect of RA on cell proliferation was abolished. Further investigation showed that treatment with 2-APB or BTP-2 inhibited RA induction of RA response element (RARE) activation in granulosa cells, confirming an important role for Ca2+signaling in mediating RA actions. Overall, these data support a model in which RA regulates ovarian follicle development by stimulating granulosa cell proliferation and that this stimulatory effect is at least in part driven by the modulation of Ca2+signaling.