The ovary can be infected by a variety of viruses, which may come from the female reproductive tract or the peritoneum. The innate immune responses to viral infection in the human ovary are poorly understood. The present study demonstrated that human ovarian granulosa cells had innate immune activity in response to viral RNA challenge through Toll-like receptor 3 (TLR3) activation. TLR3 was constitutively expressed in the human ovary and predominantly located in granulosa cells of developmental follicles at all stages. Polyinosinic-polycytidylic acid [poly (I:C)], a synthetic viral double-stranded RNA analog, induced innate immune responses in human ovarian granulosa cells and affected endocrine function. Poly (I:C) significantly upregulated proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, IL-1 beta and type I interferon (IFN-alpha/beta), and the innate immune responses were significantly reduced by blocking TLR3 signaling. Furthermore, poly (I:C) induced antiviral genes expression, including 2 '-5 '-oligoadenylate synthetase, Mx GTPase 1, IFN-stimulating gene 15 and double-stranded RNA-activated protein kinase R. In contrast, the expression of P450 aromatase and inhibin was dramatically inhibited by poly (I:C). Both silencing of TLR3 and neutralizing TNF-alpha reversed the inhibitory effect of poly (I:C) on P450 aromatase and inhibin expression. Our study demonstrates that granulosa cells play a potential role in innate immune protection against viral infection in the normal human ovary, and the innate immune response perturbs cell endocrine function.