Aim of this study was to explore the effects of cryopreservation on biological characteristics of wharton's jelly derived mesenchymal stem cells (WJ-MSC), and to provide experimental evidence for clinical applications and the establishment of WJ-MSC bank. Primary WJ-MSC were produced by umbilical cord tissue culture in vitro. Fifth passage of WJ-MSC acquired by continuous cell culture were mixed with cryoprotectants, frozen in -80°C refrigerator and stored in liquid nitrogen. After the cryopreserved WJ-MSC were thawed, the first passage of WJ-MSC was obtained through cell culture and was taken as the 1st preserved passage (PP1). Thus, PP2-PP15 WJ-MSC were obtained by continuous cell subculture. The 1st control passage (CP1) to 15th passage (CP15) represented the 6th passage to 21st passage WJ-MSC acquired by subculturing in non-cryopreserved group. The biological characteristics of WJ-MSC from cryopreserved and control group, including the recovery rate of nucleated cells, trypan blue exclusion, CCK-8 activity, cell apoptosis, cell adherence, proliferation index, cell surface antigen, cell cycle and the capacities of induced differentiation into adipocyte, osteoblast and neuron, were detected and compared. The results indicated that the recovery rate of nucleated cells of cryopreserved WJ-MSC was 98.2%, trypan blue exclusion rate was 94.3%, CCK-8 activity was 91.4%, apoptotic rate was 3.9%, and the adherence rate was 92.6%. There was a statistically significant difference in proliferation index between PP1 and CP1 (P < 0.05), but there were no statistically significant differences between PP2-PP15 and their corresponding controls. The subculture cells highly expressed CD29, CD44, CD71, CD73, CD90, CD105, CD166 and HLA-ABC, and lowly expressed CD34, CD45 and HLA-DR. The expressions of above-mentioned surface antigens were not different statistically between two groups. The proliferation latency and logarithm proliferation of the subculture cells between two groups were also not different. After induced differentiation into adipocyte, osteoblast and neuron, the staining with oil red O, alkaline phosphatase and neuron-specific enolase was performed respectively, and the positive degrees were not clearly different macroscopically between two groups. Relatively high levels of triglyceride, alkaline phosphatase, and neuron-specific enolase in relevant cells could be detected, but had no significant differences between two groups. It is concluded that some WJ-MSC (< 10%) are damaged after cryopreservation, and the biological characteristics of WJ-MSC in cryopreservation group keep constant, as compared with that in non-cryopreservation group.