Background: Sodium nitroprusside (SNP) releases nitric oxide which has signaling role. Objectives: This study was conducted to understand the role of low concentration of SNP on viability, proliferation and biochemical properties of rat bone marrow mesenchymal stem cells (MSCs). Materials and Methods: MSCs were used to evaluate the viability and morphology in presence of SNP (1 to 100 μM) at 12, 24 and 36 hours. Then 10, 50 and 100 μM of SNP as well as 24 hours were selected for further study. Cell proliferation was investigated by colony forming assay and population doubling number (PDN). Calcium (Ca2+) potassium (K+) and sodium (Na+) level as well as activity of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were measured. Results: The MSCs viability increased when treatment with 1 and 10 μM at all the treatment periods while 90 and 100 μM caused significant reduction after 24 and 36 hours. Also 10 μM caused elevation whereas 50 and 100 μM showed reduction of proliferation ability. We observed morphological changes and significant reduction of all the investigated enzymes with 100 μM. Activity of ALT and AST were elevated with 10 μM after 24 hours, whereas LDH and ALP activities were not changed. Na+, K+ and Ca2+ was not changed due to 10 and 50 μM treatments, whereas 100 μM only elevated the level of calcium and sodium ions. Conclusions: Low concentration of SNP caused increase of viability and proliferation due to metabolic activity elevation. But the high concentration of SNP induced cell viability and proliferation reduction caused by metabolic and ionic imbalance as well as infrastructure alteration.