Integrins play a pivotal role in proliferation, differentiation and survival in skeletal and cardiac myocytes. The 1D isoform of the 1 integrin is specifically expressed in striated skeletal muscle. However, little is known about the role and the mechanisms by which the splice variant 1D integrin regulates myogenesis and mechanotransduction. We observed that cyclic mechanical stretch increases 1D integrin protein levels and activates the downstream cytoskeletal signaling proteins, FAK and RhoA. Elimination of native 1D integrin expression by RNAi in immature developing myoblasts abolished stretch-induced increases in FAK phosphorylation and further down-regulated RhoA activity. The blocking of 1D integrin expression prevented myocellular fusion to form multinucleated mature myotubes. Restoration of human 1D integrin expression in 1D integrin deficient partially restored myotube formation. The onset of myofusion also requires the generation of nitric oxide (NO). The release of NO affects cytoskeletal proteins by mediating RhoA activity and protein degradation. Our previous study demonstrated that stretched-induced NO positively modulates mechanical properties of differentiating skeletal myocytes. We found a significant decrease in NO production and apparent elastic modulus in 1D integrin deficient cells, suggesting signaling interactions between 1D integrin and nNOS to mediate mechanotransduction and myogenesis in skeletal myocytes. These results suggest that, in addition to regulating differentiation, the 1D integrin isoform plays a critical role in the response of skeletal myoblasts to cyclic stretch by activating the downstream components of FAK and RhoA activity and affecting NO release.