Background: Multiple signaling pathways have been found to regulate the osteoblast differentiation of human bone marrow stromal (skeletal) stem cells (hBMSCs) including ROCK signaling pathway. ROCK1 and ROCK2 are implicated in various cellular activities involving cellular proliferation, apoptosis, cell adhesion, motility, extracellular matrix remodeling, actin cytoskeleton organization, and contraction of smooth muscles. However, the crucial role of ROCK signaling during osteoblast differentiation of hBMSCs, is not studied in details. Methods: A ROCK1/2 inhibitor, GSK429286A, identified during a chemical biology screen of a small molecule library for their effect on osteoblast differentiation of hBMSCs. Alkaline phosphatase activity and staining tests, indicators for osteoblastic differentiation and Alizarin red staining, indicator for in vitro formation of mineralized matrix, were performed. Changes in gene expression were assessed using qRT-PCR. Results: In vitro treatment of hMSCs with GSK429286A (3µM) led to significant increase in osteoblast differentiation as demonstrated by elevated activity of the ALP, formation of mineralized matrix in vitro and upregulation of osteoblast-related expression of genes including ALP, OC, ON, and OPN. To determine the molecular mechanisms, we examined the effects of GSK429286A on a molecular signature of a number of gene targets known to affect hBMSCs differentiation into osteoblasts. GSK429286A upregulated gene expression of LIF, SOCS3, RRAD, NOTCH3, TNF, CXCL1, VDR, IL6, and THBS2, which are targets of a number of signaling pathways e.g. TGFβ, insulin, focal adhesion, Notch, Vitamin D and cytokine signaling. Conclusions: We identified a ROCK1/2 inhibitor (GSK429286A) as a positive regulator of hBMSC osteoblastic distinction which could be beneficial as a therapeutic alternative for managing bones diseases characterized by impaired bone formation.