Muscle fibers are generally formed as multinucleated fibers that are differentiated from myoblasts. Several reports have identified transcription factors and proteins involved in the process of muscle differentiation, but the roles of microRNAs (miRNAs) in myogenesis remain unclear. Here, comparative analysis of the miRNA expression profiles in mouse myoblasts and gastrocnemius (GA) muscle uncovered miR-3074-3p as a novel miRNA showing markedly reduced expression in fully differentiated adult skeletal muscle. Interestingly, elevating miR-3074-3p promoted myogenesis in C2C12 cells, primary myoblasts, and HSMMs, resulting in increased mRNA expression of myogenic makers such as Myog and MyHC. Using a target prediction program, we identified Caveolin-1 (Cav1) as a target mRNA of miR-3074-3p and verified that miR-3074-3p directly interacts with the 3′ untranslated region (UTR) of Cav1 mRNA. Consistent with the findings in miR-3074-3p-overexpressing myoblasts, knockdown of Cav1 promoted myogenesis in C2C12 cells and HSMMs. Taken together, our results suggest that miR-3074-3p acts a positive regulator of myogenic differentiation by targeting Cav1.