Myogenesis is a multistep developmental program that generates and regenerates skeletal muscles. Several extracellular factors have been identified that participate in the regulation of myogenesis. Although skeletal muscles are always subjected to mechanical stress in vivo, the role of mechanical forces in the regulation of myogenesis remains unknown. We have investigated the molecular mechanisms by which cyclic mechanical strain modulates myogenesis. Application of cyclic mechanical strain using the computer-controlled Flexcell Strain Unit increased the proliferation of C2C12 cells and inhibited their differentiation into myotubes. Cyclic strain increased the activity of cyclin-dependent kinase 2 (cdk2) and the cellular level of cyclin A, and inhibited the expression of myosin heavy chain and formation of myotubes in C2C12 cultures. The activity of nuclear factor-kappa B (NF-kappaB) transcription factor and the expression of NF-kappaB-regulated genes, cyclin D1 and IL-6, were augmented in response to mechanical strain. Cyclic strain also increased the activity of Rho GTPases, especially Rac-1. The inhibition of Rho GTPases activity, by overexpression of Rho GDP dissociation inhibitor (Rho-GDI), inhibited the strain-induced activation of NF-kappaB in C2C12 cells. Overexpression of either NF-kappaB inhibitory protein IkappaBalphaDeltaN (a degradation resistant mutant IkappaBalpha) or Rho-GDI blocked the strain-induced proliferation of C2C12 cells. Furthermore, overexpression of FRNK, a dominant negative mutant of focal adhesion kinase (FAK), inhibited the strain-induced proliferation of C2C12 cells. Our study demonstrates that cyclic mechanical strain inhibits myogenesis through the activation of FAK, Rac-1, and NF-kappaB.