In this study, Squalene epoxidase (SQLE) overexpression vector was transfected into bovine skeletal muscle-derived mesenchymal stem/stromal cells (MSCs) to study the molecular mechanism of SQLE regulating meat quality through myogenesis. We initially profiled the expression of SQLE in cattle embryos and adults, in the muscle tissue of four different cattle varieties, and in 11 different tissues/organs of Guangxi cattle variety. Subsequently, we isolated and cultured bovine skeletal muscle-derived MSCs and detected the expression of SQLE during cell proliferation and differentiation. Then, we constructed a bovine SQLE overexpression vector and transfected it into bovine skeletal muscle-derived MSCs by liposome transfection. Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), flow cytometry, immunofluorescence, and quantitative polymerase chain reaction assays were used to characterize cell proliferation and differentiation in detail. The results showed that the relative expression level of bovine SQLE gene in brain tissue was the highest, and in adult muscle tissue was significantly higher than that in embryonic stage. Especially, the expression of SQLE was significantly upregulated in cell differentiation stage. Furthermore, the proliferation, cell cycle, apoptosis, and myoblast differentiation assays indicated that SQLE significantly promoted the differentiation and apoptosis of bovine skeletal muscle-derived MSCs, but inhibited their proliferation. In conclusion, our study reveals the role of SQLE in myoblast differentiation. These results will provide new clues for the regulation network of bovine muscle development.