Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1(KD) cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1(KD) cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1(KD) cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1(KD) cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell-cell fusion. Toca-1(KD) cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASP(KO) MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1(KD) cells restored myotube formation of Toca-1(KD) cells. Thus, our results suggest that Toca-1(KD) cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.