Calcium-dependent chloride conductances have been described in chicken and human granulosa cells (GCs) and may be involved in steroidogenesis. However, the molecular identities of corresponding chloride channels remain unknown. The purpose of this study was to explore the expression and function of the Anoctamin 1 (ANO1) calcium-activated chloride channel (CaCC) in mouse ovary. ANO1 mRNA and protein expression was identified in mouse ovary GCs by RT-PCR, immunoblot, and immunostaining. Patch-clamp analysis on freshly isolated GCs identified an outwardly rectifying Ca(2+)-activated Cl(-) current that was completely blocked by a selective ANO1 inhibitor T16Ainh-A01. Knockdown of ANO1 mRNA or incubation with a selective inhibitor T16Ainh-A01 enhanced estradiol production, whereas a selective ANO1 activator Eact significantly inhibited estradiol production in primary cultured GCs. The ANO1 expression or activation increases the phosphorylation of ERK1/2 and decreases aromatase expression. The ANO1 expression level is remarkably higher at the proestrous and estrous stages in the estrous cycle. In vivo study indicated a profound induction of ANO1 expression in ovarian GCs by pregnant mare serum gonadotropin (PMSG) that can be further augmented by hCG treatment, suggesting that both FSH and LH may upregulate ANO1 expression at the proestrous and estrous stages. ANO1 expression was remarkably reduced in DHEA-induced PCOS ovary. These data identified for the first time the expression of ANO1 Ca(2+) activated Cl(-) channel in mouse ovarian GCs and determined its negative regulation on estrogen production possibly through MEK-ERK signaling cascade. The present study provided new insights into the molecular mechanisms for the regulation of folliculogenesis and ovulation.