Abstract: Objective To study the role of Wnt/β-catenin signaling pathway in regulating the differentiation of muscle derived stem cells (MDSC). Methods MDSC cultured in vitro served as a control group, MDSC induced by valproic acid (VA) to differentiate into neural-like cells served as an experimental group, and MDSC induced by VA and DKK-1 served as an inhibitor group in this study. The Wnt/β-catenin signaling pathway was inhibited with sFRP. The differentiation of MDSC was detected with immunocytochemistry staining. The expression of Wnt3a mRNA, GSK-3β and β-catenin in MDSC was detected by RT-PCR and Western blot, respectively. Results The VA-induced MDSC differentiated into neural-like cells in experimental group and inhibitor group. The differentiation rate was significantly lower in inhibitor group than in experimental group. No MDSC were differentiated in control group. The expression level of Wnt3a mRNA and β-catenin was significantly higher in experimental group and inhibitor group than in control group and in experimental group than in inhibitor group. The expression level of GSK-3β was significantly lower in experimental group and inhibitor group than in control group. Conclusion The Wnt/β-catenin signaling pathway is activated when MDSC are differentiated into neural-like cells, and blocking the Wnt/β-catenin signaling pathway can thus inhibit the differentiation of MDSC into neural-like cells.