The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10µg/l) and basic fibroblastic growth factor (bFGF, 10µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20±8.677 (positive ratio 0.2140±0.0119) to 109.20±8.430 (positive ratio 0.3193±0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30±8.486 (positive ratio 0.18±0.0152) to 107.50±8.683 (positive ratio 0.27±0.0115) (P<0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74±0.05 vs. 1.03±0.06; GFAP: 0.64±0.08 vs. 0.97±0.05, both P<0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases.