Abstract: Objective To investigate the differentiation inducing effect of 5-Azacytidine (5-Aza) in rat adipose-derived mesenchymal stem cells (ADMSCs) and the expressions of related protein. Methods The abdominal subcutaneous adipose tissue of Sprague-Dawley (SD) rat was isolated and cultured by enzyme digestion and adhesion method. The cultured cells were identified by using anti-monocyte human leukocyte antigen DR (HLA-DR), rabbit anti-CD13, CD44, CD105 and von willebrand factor (vWF) polyclonal antibody. The hematopoietic stem cells and fibroblasts were excluded. The third passage ADMSCs cells were used to inoculate into 24 well culture plates with the concentration of 1×10⁴ cells/well. And the cultured cells were randomly divided into normal group and induction group, the cells in normal group was cultured in normal saline with sterile saline, the cells in induction group was incubated with 5-Aza at a concentration of 10 μmol/L. The morphological changes, cardiac troponin T (cTnT), cardiac troponin I (cTnI) and Connexin43 mRNA expression and cTnT, cTnI, Connexin43 protein expression were compared between the two groups. Results The proliferation of ADMSCs cells in the induction group was slowly, their volume was increased and gradually changed into a rod-shaped or wide-spindle shape, the morphology was characterized by cardiomyocytes; RT-qPCR results showed that the mRNA expression of cTnT, cTnI and Connexin43 were enhanced in the induced group; Immunohistochemistry results showed that the brownish yellow or chocolate brown granular matters were obviously visible in the cytoplasm of induction group, there were fewer brownish yellow or chocolate brown granular matters in the cytoplasm of the normal group, and the protein positive expression scores of cTnT, cTnI and Connexin43 in the induced group was significantly higher than that of the normal group (P<0.05). Conclusion Rat adipose tissue contains mesenchymal stem cells (MSCs) with multi-directional differentiation potential, 5-Aza can induce rat ADMSCs to differentiate into cardiomyocytes cells, and ADMSCs are expected to become the seed cells for the treatment of myocardial infarction.