Muscle regeneration is essential for proper muscle homeostasis and relies primarily on muscle stem cells (MuSC). MuSC are maintained quiescent in their niche and can be activated following muscle injury. Using an in vitro model of primary human quiescent MuSC (called reserve cells, RC), we analyzed their Ca2+ response following their activation by fetal calf serum and assessed the role of Ca2+ in the processes of RC activation and migration. The results showed that RC displayed a high response heterogeneity in a cell-dependent manner following serum stimulation. Most of these responses relied on inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release associated with Ca2+ influx, partly due to store-operated calcium entry. Our study further found that blocking the IP3 production, Ca2+ influx, or both did not prevent the activation of RC. Intra-or extracellular Ca2+ chelation did not impede RC activation. However, their migration potential depended on Ca2+ responses displayed upon stimulation, and Ca2+ blockers inhibited their movement. We conclude that the two major steps of muscle regeneration, namely the activation and migration of MuSC, differently rely on Ca2+ signals.