Mechanically activated (MA) Piezo1 channels play an important role in both normal physiology and pathological dysfunction in multiple tissues and organs. In skeletal muscle cells, Piezo1 channels are involved in the regulation of postnatal myogenesis and muscle regeneration after injury. To further understand the role of MA Piezo1 channels as potential critical sensors of mechanical perturbations during muscle contractions, we studied the possible contribution of MA Piezo1 channels to enhanced protein synthetic response of C2C12 myotubes to mechanical simulation. C2C12 myotubes were subjected to mechanical stimulation by electrical pulse stimulation (EPS) alone or EPS in combination with Yoda1, Gadolinium or Yoda1 + Gadolinium. EPS alone elicited an increase in anabolic signaling and protein synthesis (PS). Incubation with Yoda1 during EPS enhanced anabolic signaling and PS compared to EPS alone. Gadolinium or Yoda1 + gadolinium during EPS abolished or diminished the Yoda1 + EPS-induced effects on anabolic signaling and PS. Our work demonstrates that chemical activation of Piezo1 channels during mechanical stimulation contributes to enhanced protein anabolism in C2C12 myotubes.