Mesencymal stem cells have been used as an “off the shelf” cellular therapeutics due to their immunomodulatory properties. However, cryoprotectants used to store these cells have not been adequately studied, in terms of their safety and efficacy. In this study, we investigated whether use of natural cryoprotectants free of animal products would be effective in the cryopreservation of human bone marrow-derived mesenchymal stem cells (BMSCs). Cryopreservation medium containing disaccharides, antioxidants and caspase inhibitors combined with a reduced concentration of dimethylsulfoxide (DMSO) was used to cryopreserve BMSCs. The cells were tested for viability with cck-8 assay and a growth curve was generated to measure population doubling. In addition, flow cytometry analysis for cell surface antigens, and RT-PCR for mRNA expression of stem cell markers were performed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) DMSO produced results similar to those for that of the control (10% (v/v) DMSO and 30% FBS), in terms of cell viability, culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in BMSCs cryopreserved for 3 weeks. Thus, these results show that BMSCs can be cryopreserved with a reduced concentration of DMSO with the addition of disaccharides, antioxidants and caspase inhibitors. This study suggests that human BMSCs can be effectively cryopreserved using natural cryoprotectants combined with reduced concentration of DMSO for cell therapy applications.