Signal transduction cascades involving Rho-associated kinases (ROCK), the serine/threonine kinases downstream effectors of Rho, have been implicated in the regulation of diverse cellular functions including cytoskeletal organization, cell size control, modulation of gene expression, differentiation, and transformation. Here we show that ROCK2, the predominant ROCK isoform in skeletal muscle, is progressively up-regulated during mouse myoblast differentiation and is highly expressed in the dermomyotome and muscle precursor cells of mouse embryos. We identify a novel and evolutionarily conserved ROCK2 splicing variant, ROCK2m, that is preferentially expressed in skeletal muscle and strongly up-regulated during in vivo and in vitro differentiation processes. The specific knockdown of ROCK2 or ROCK2m expression in C2C12 myogenic cells caused a significant and selective impairment of the expression of desmin and of the myogenic regulatory factors Mrf4 and MyoD. We demonstrate that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42 and p44 mitogen-activated protein kinase-p90 ribosomal S6 kinase-eucaryotic elongation factor 2 intracellular signaling pathways and, thereby, positively regulate the hypertrophic effect elicited by insulin-like growth factor 1 and insulin, linking the multifactorial functions of ROCK to an important control of the myogenic maturation.