In this study, we used a chitosan hydrogel as a 3-dimensional substrate for the attachment, proliferation, and differentiation of rat muscle-derived stem cells (rMDSCs) in the presence of valproic acid (VA). Chitosan solutions containing glycerol phosphate disodium salt form a hydrogel at body temperature. The chitosan hydrogel exhibited a porous 3-dimensional network that allowed the culture medium to penetrate. The chitosan hydrogel acted as a suitable biocompatible substrate for the attachment and proliferation of rMDSCs. On chitosan hydrogel in the presence of VA, rMDSCs exhibited higher expression of the neural markers, neuron-specific enolase (NSE) and beta tubulin III (Tuj-1), the oligodendrocyte marker, oligodendrocyte transcription factor 2 (Olig-2), and the astrocyte marker, glial fibrillary acidic protein (GFAP) than those in the absence of VA. Our results suggest that rMDSCs on a chitosan hydrogel in the presence of VA can differentiate into cells with a neural-like phenotype.