The proliferation and differentiation of skeletal muscle satellite cells is a complex physiological process involving various transcription factors and small RNA molecules. This study aimed to understand the regulatory mechanisms underlying these processes, focusing on interferon-related development factor 2 (IFRD2) as a target gene of miRNA-2400 in bovine skeletal MuSCs (MuSCs). IFRD2 was identified as a target gene of miRNA-2400 involved in regulating the proliferation and differentiation of bovine skeletal MuSCs. Our results indicate that miR-2400 can target binding the 3'UTR of IFRD2 and inhibit its translation. mRNA and protein expression levels of IFRD2 increased significantly with increasing days of differentiation. Moreover, overexpression of the IFRD2 gene inhibited proliferation and promoted differentiation of bovine MuSCs. Conversely, the knockdown of the gene had the opposite effect. Overexpression of IFRD2 resulted in the inhibition of ERK1/2 phosphorylation levels in bovine MuSCs, which in turn promoted differentiation. In summary, IFRD2, as a target gene of miR-2400, crucially affects bovine skeletal muscle proliferation and differentiation by precisely regulating ERK1/2 phosphorylation.