The regulatory roles of the non-neuronal cholinergic system, such as its growth promoting effects on mural granulosa cells during ovarian folliculogenesis, have been reported for several mammalian species; however, its roles in the early stages of follicles are not well-understood. This study was conducted to examine the expression of transcripts involved in the non-neuronal cholinergic system, and the effects of activating acetylcholine (ACh) signaling in mouse ovaries. The transcripts encoding proteins required in processes critical for ACh metabolism and a nicotinic cholinergic receptor, CHRNA7, were detected in ovaries and isolated mural granulosa cells. Stimulation with carbachol, a cholinergic agonist, promoted proliferation of mural granulosa cells in vitro. Moreover, carbachol treatment of neonatal ovaries significantly increased the numbers of primordial follicles compared with control untreated ovaries in organ cultures. These results suggest that a functional non-neuronal cholinergic system exists in mouse ovaries, and that it promotes the proliferation of mural granulosa cells and the formation/survival of primordial follicles.