MicroRNA (miRNA)s are a class of non‐coding RNAs that regulate gene expression post‐transcriptionally. Muscle‐specific miRNA, miRNA (miR)‐1, miR‐133 and miR‐206 play a crucial role in the regulation of muscle development and homeostasis. Muscle injuries are a common muscloskeletal disorder, and the most effective treatment has not been established yet. The purpose of this study was to demonstrate that a local injection of double‐stranded (ds) miR‐1, miR‐133 and 206 can accelerate muscle regeneration in a rat skeletal muscle injury model. After the laceration of the rat tibialis anterior muscle, ds miR‐1, 133 and 206 mixture mediated atelocollagen was injected into the injured site. The control group was injected with control siRNA. At 1 week after injury, an injection of miRNAs could enhance muscle regeneration morphologically and physiologically, and prevent fibrosis effectively compared to the control siRNA. Administration of exogenous miR‐1, 133 and 206 can induce expression of myogenic markers, MyoD1, myogenin and Pax7 in mRNA and expression in the protein level at 3 and 7 days after injury. The combination of miR‐1, 133 and 206 can promote myotube differentiation, and the expression of MyoD1, myogenin and Pax7 were up‐regulated in C2C12 cells in vitro. Local injection of miR‐1, 133 and 206 could be a novel therapeutic strategy in the treatment of skeletal muscle injury.