ABSTRACT Background: Sodium arsenite (SA) recently has been recommended to be used in malignancy therapy. Our studies showed, SA in short and long period of treatment caused reduction of rats Bone Marrow Mesenchymal Stem Cells (MSCs) viability and induced caspase dependent apoptosis. The aim of this study was to investigate the effect of SA on osteogenic differentiation of MSCs. Methods: MSCs were extracted and expanded to third passage, then cultured in DMEM supplemented with osteogenic media in presence of 1 and 25nM of SA for 21 days. The viability and the level of mineralization were determined using MTT assay and alizarin red respectively. In addition morphology and nuclear diameter of the cells were studied with the help of fluorescent dye. Furthermore, calcium content and alkalinphosphatase activity also were estimated using commercial kit. Data was statistically analyzed and the P<0.05 was taken as the level of significant. Results: The viability and mineralization of the cells treated with SA reduced significantly (P<0.05) after tenth day in compare with control. Also, chromatin condensation, reduction of nuclei diameter and cytoplasm shrinkage were observed in the cell treated with 1 and 25 nM concentrations. The calcium and alkalinphosphatase activity of the cells decreased significantly with 1 and 25 nM concentrations of SA when compared with control. Conclusion: Adverse effect of SA was observed on osteogenic differentiation of MSCs at 1 and 25 nM due to disruption of mineralization. We strongly suggest more investigation to be run on this chemical with respect to the therapy of the malignant patients.