Myocytes withdraw from the cell cycle to differentiate during muscle development. Given the capacity of microRNAs (miRNAs) to regulate gene expression during development, we screened for miRNAs that were associated with muscle development. S-Poly(T) Plus analysis of 273 miRNAs in porcine longissimus dorsi muscles revealed 14 miRNAs that were strongly upregulated with age of postnatal muscle development , including miR-195 and miR-497. These two miRNAs were also strongly upregulated at late differentiation stages of mouse skeletal myoblast C2C12 cells, and demethylation treatment induced significant upregulation of miR-195/497. Manipulation of miR-195/497 expression resulted in dramatic changes in the proliferation and differentiation of C2C12 cells. We identified high-mobility group AT-hook 1 ( ) mRNA as a highly conserved target of miR-195/497 in C2C12 myoblasts. Overexpression of miR-195/497 or silencing in C2C12 cells promoted myogenic differentiation. Moreover, we showed that miR-195/497 repressed , which in turn downregulated one of the HMGA1 downstream targets , whose inhibitory effect on myogenic differentiation is well established. Our study revealed a subset of potential development-associated miRNAs and suggests a novel regulatory axis for myogenesis in which miR-195/497 promote myogenic differentiation by repressing the HMGA1-Id3 pathway.