Introduction: Diethylhexyl phthalate (DEHP) is leaching form polyvinyl chloride to cause animal toxicity. In our previous study, DEHP caused osteoblasts mortality in vitro, since rat bone marrow mesenchymal stem cells (MSCs) is the cellular back up for osteoblasts; therefore its effect on MSCs was investigated. Methods: MSCs were extracted from Westar rats and treated with 0.5 to 2500μM of DEHP for 12, 24 and 48h to study the viability. Then further investigations, including proliferation, cell morphology, sodium and potassium level, concentration of calcium, total protein, activity of metabolic enzymes (ALT, AST, ALP, LDH), malondialdehyde (MDA) level, total antioxidant capacity, activity of superoxide dismutase (SOD) and catalase (CAT) were measured using selected concentration (100, 500 and 1500μM). Results: The 100μM of DEHP did not change the viability and biochemical factor after 48h but colony forming assay and population doubling number was significantly affected. Th 500μM only reduced the viability at 24 and 48h, while 1500μM caused the significant reduction at all the periods. These two concentrations, caused significant proliferation reduction as well as significant increase in calcium and sodium level, LDH activity and MDA level. In addition, we observed decrease in potassium, total protein, activity of metabolic enzymes and activity of CAT and SOD significantly. Conclusion: DEHP has reduced viability and proliferation of MSCs through metabolic change, alteration in cellular ultrastructure, ionic imbalance and induction of oxidative stress.