Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific overexpression of constitutively activated Notch1 Intracellular Domain (NICD(OE)), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independent of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression, thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effect of NICD(OE) on MyoD and Ki67, but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ that binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through directly regulating Pax7.