MicroRNAs are evolutionarily conserved small RNAs that post-transcriptionally regulate gene expression and have emerged as critical regulators of skeletal muscle development. Here, we identified miR-148a as a novel myogenic microRNA that mediated myogenic differentiation. The expression levels of miR-148a increased during C2C12 myoblast differentiation. Overexpression of miR-148a significantly promoted myogenic differentiation of both C2C12 myoblast and primary muscle cells. Blocking the function of miR-148a with a 2'-O-methylated antisense oligonucleotide inhibitor repressed C2C12 myoblast differentiation. Using a bioinformatics approach, we identified Rho-associated coiled-coil containing protein kinase 1 (ROCK1), a known inhibitor of myogenesis, as a target of miR-148a. A dual-luciferase reporter assay was used to demonstrate that miR-148a directly targeted the 3'-UTR of ROCK1. In addition, the overexpression of miR-148a decreased the protein expression of ROCK1 in C2C12 myoblast and primary muscle cells. Furthermore, ROCK1 inhibition with specific siRNA leaded to accelerated myogenic differentiation progression, underscoring a negative regulatory function of ROCK1 in myogenesis. Therefore, our results revealed a novel mechanism in which miR-148a positively regulates myogenic differentiation via ROCK1 down-regulation.