The mammary gland is a dynamic organ that undergoes significant changes at multiple stages of postnatal development. Although the roles of systemic hormones and microenvironmental cues in mammary homeostasis have been extensively studied, the influence of neural signals, particularly those from the sympathetic nervous system, remains poorly understood. Here, using a mouse mammary gland model, we delved into the regulatory role of sympathetic nervous signaling in the context of mammary stem cells and mammary development. Our findings revealed that depletion of sympathetic nerve signals results in defective mammary development during puberty, adulthood, and pregnancy, accompanied by a reduction in mammary stem cell numbers. Through in vitro three-dimensional culture and in vivo transplantation analyses, we demonstrated that the absence of sympathetic nerve signals hinders mammary stem cell self-renewal and regeneration, while activation of sympathetic nervous signaling promotes these capacities. Mechanistically, sympathetic nerve signals orchestrate mammary stem cell activity and mammary development through the extracellular signal-regulated kinase signaling pathway. Collectively, our study unveils the crucial roles of sympathetic nerve signals in sustaining mammary development and regulating mammary stem cell activity, offering a novel perspective on the involvement of the nervous system in modulating adult stem cell function and organ development.