Objective MicroRNAs (miRNAs) are highly conserved, endogenous small RNAs that regulate gene expression at the post-transcriptional level. miR-127 plays an essential role in myogenic differentiation in vivo and in vitro. However, it is not clear whether miR-127-3p affects myogenic cell proliferation. Methods The detailed function of miR-127-3p in proliferative C2C12 cell lines and further identified its regulatory mechanism by qRT-PCR, western blot, flow cytometry analysis and luciferase reporter assay. Results Overexpression of miR-127-3p significantly inhibited proliferation of C2C12 cells and vice versa. Sept7 was a target gene of miR-127-3p using dual-luciferase reporter assay, qRT-PCR, and western blotting. The RNA interference analysis, in which Sept7 was downregulated, showed that Sept7 significantly promoted the proliferation of C2C12 cells. Besides, the expression level of Sept7 was detected analysis in muscle cells and tissues. Conclusions These findings reveal that miR-127-3p regulates myoblast proliferation by targeting Sept7.