Abstract Differentiation of human mesenchymal stem cells (MSCs) to metabolically active hepatocytes depends on different regulatory factors. Trans-differentiation of stem cells into specific cell lineage in presence of specific stimuli is associated with the molecular and cellular damages. The aim of the present study was to examine the role of P53 in regulation of cyclooxygenase-2 (COX-2) expression and generation of protein and lipid oxidation during trans-differentiation of MSCs into hepatocyte-like cells. During the 3-weeks differentiation process of MSCs to hepatocyte-like cells we found that expressing liver-specific markers was associated with increased levels of lipid peroxidation and protein carbonyl formation. Expression of P53 and COX-2 expression at mRNA and protein levels were evaluated in MSCs before and after differentiation on days 7, 14 and 21. We showed that the upregulation of COX-2 was associated with augmentation of the rate of cell proliferation, morphological and biochemical changes of hepatocytes-like cells. However, in parallel the P53 at the mRNA level was down regulated and in protein levels accumulation in the nuclei was reduced during the hepatogenic differentiation time. Our results may suggest a P53-COX-2 pathway in regulation of hepatogenic differentiation of stem cells which is linked to differentiation dependent molecular oxidative damage.