Though miRNAs have been reported to regulate bovine myoblast proliferation, but many miRNAs still need to be further explored. Specifically, miR-152 is a highly expressed miRNA in cattle skeletal muscle tissues, but its function in skeletal muscle development is unknown. Herein, we aimed to investigate the role of miR-152 in regulating bovine myoblast proliferation. Functionally, RT-qPCR, Western blotting, EdU assay, and flow cytometry detection results showed that miR-152 inhibited bovine myoblast proliferation. Mechanistically, we demonstrated transcription factor KLF6 was a target gene of miR-152 by means of bioinformatics software prediction and dual-luciferase report analysis, which had been demonstrated to be favorable for myoblast proliferation. Collectively, our research suggested that miR-152 inhibits bovine myoblast proliferation via targeting KLF6.