Objective To explore the effect of cryopreservation on the biological and differentiation potential of mouse adipose-derived mesenchymal stem cells (ADMSCs)in vitro. Methods ADMSCs were isolated from mice fatty tissue and then cultured. The ADMSCs of third passage were cryopreserved in liquid nitrogen (-196℃) for 12 months as the experimental group. After recovery, a MTT method was applied to get ADMSCs growth curve. SA-β-Gal staining was used to detect ADMSCs senescence. Immunocytochemical method was used to detect the expression of cell surface molecules, such as CD73, CD90 and CD105. After induction, immunofluorescence was to applied to detect the expression of cardiac-specific troponin (cTnT). Alizarin red, alkaline phosphatase and oil red O stainings were used to detect the differentiation potential to osteogenic and adipogenic, respectively. Real time-PCR was used to detect the mRNA expression of cTnT, Gata4, Ost and Runx2. ADMSCs without cryopreservation were used as the control group. Results ADMSCs growth and senescence index were no significant difference between cryopreservation group and non-cryopreservation group (P>0.05). After induction, there was no significant difference in the expression of cTnT and the positive reaction of alizarin red, alkaline phosphatase and oil red O staining between two groups(P>0.05). Conclusion Cryopreservation has no significant effects on the biological characteristics of ADMSCs in vitro, including the basic biological properties and differentiation potential into cardiomyocytes, adipocytes and osteoblasts.