Muscle stem cells (MuSCs) represent a promising starting material for the production of cultured meat. However, MuSCs exhibit impaired proliferative capabilities when cultured at high-density, with the underlying signaling pathways yet to be fully characterized. In this study, we revealed that Notch signaling was activated in response to high-density conditions in porcine MuSCs. Consistently, treatment with DAPT, a specific inhibitor of Notch signaling, significantly improved the proliferation of MuSCs cultivated at high-density. Further, Notch signaling was gradually repressed during MuSC differentiation. Notably, DAPT accelerated this downregulation process, thereby significantly promoting the myogenic differentiation potential of MuSCs. In summary, this study highlights the critical role of Notch signaling in regulating the proliferation and differentiation of MuSCs. Through modulating the activity of Notch signaling, we have identified a strategy to achieve extensive expansion of MuSCs and to enhance their myogenic differentiation.