Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering. It is well known that contact guidance by grooves/ridges structures induces myoblasts to align and to migrate along the anisotropic direction. In this study, two series of grooved substrata with different widths (450 and 900 nm) and different depths (100, 350, and 550 nm) were studied on their effects on myoblast adhesion, proliferation, and differentiation into myotubes. We found that C2C12 cells were aligned and elongated along the direction of grooves. Groove depth was more influential on cellular morphology, proliferation, and differentiation than groove width. While cell proliferation was retarded on the grooved surfaces especially on the substrate with 900/550 nm (width/depth), differentiation was also enhanced on the patterned surfaces compared to the flat control. Our results demonstrated the potential of grooved substrata with submicron scale in skeletal muscle tissue engineering.