Dimethyl sulfoxide (DMSO), a commonly used cryoprotectant in cryopreservation procedures, is detrimental to viability of cells. In this view point, a comparative study was carried out to evaluate the effect of DMSO on porcine mesenchymal stem cells (pMSCs). We compared the viability, colony forming unit-fibroblast (CFU-F) assay, expression of Bak and Bcl2 genes, Bcl2 protein antigen, and CD90 in pMSCs cryopreserved with 5%, 10%, and 20% DMSO. pMSCs isolated from bone marrow were characterized by alkaline phosphatase activity and the expression of transcription factors, such as Oct 3/4, Nanog, and Sox2. The cells were then cryopreserved by cooling at a rate of -1°C/min in a programmable freezer and stored in liquid nitrogen. The results of survival of pMSCs cryopreserved at 5% DMSO were comparable to control group (fresh pMSCs). The survival and the number of colonies formed in cryopreserved pMSCs were inversely proportional to the concentration of DMSO. The number of colonies formed in pMSCs cryopreserved with all concentrations of DMSO was significantly (p < 0.05) lower than the control group. An increased tendency for Bak and Bcl2 gene expression was noticed in cryopreserved pMSCs at 3 h postthawing compared to control group. There was a close resemblance in higher level of expression of CD90 between control and cryopreserved pMSCs. Because there was no considerable difference in the results of pMSCs cryopreserved at 5% and 10% DMSO, this study strongly suggests the use of 5% DMSO in cryopreservation of pMSCs as an alternative to conventional 10% DMSO.