Objective To study the effect of cryopreservation on some biological properties of rabbit adipose-derived mesenchymal stem cells (rADMSCs). Methods rADMSCs culture was isolated by tissue explants adherent method. Morphology of the primary cells was observed by inverted microscopy. Immunophenotypes of the rADMSCs were determined using flow cytometry. The third passage cells were preserved in liquid nitrogen for 6 months, and then were thawed, and subcultured to passage 7. The growth curves of the cryopreserved cells were analyzed by MTT assay, and the cryopreserved cells were cultured in adipogenic and osteogenic medium, with non-cryopreserved rADMSCs as a control group. The adipogenic and osteogenic abilities of the rADMSCs were evaluated by oil red O staining, alizarin red staining and alkaline phosphatase activity assay, respectively. Results The rADMSCs cultured in vitro exhibited a spindle-shaped appearance and rapid growth expansion. Flow cytometry analysis revealed that the third passage rADMSCs were CD44-and CD90-positive, but negative for hematopoietic cells surface marker CD45. The growth curves of cells in the experimental and control groups were "S" shaped, showing a non-significant difference between the two groups (P>0.05). The oil red O staining and alizarin red staining results were positive at 2 weeks after adipogenic and osteogenic induction. The ALP activities of the two groups were increased with osteogenic induction time, with a non-significant difference (P> 0.05). Conclusions Cryopreservation does not affect the growth and differentiation pluripotency of rADMSCs significantly.