Previous reports have demonstrated that Reversine can reverse differentiation of lineage-committed cells to mesenchymal stem cells and suppress tumors growth. However, the molecular mechanisms of antitumor activity and promoting cellular dedifferentiation for reversine have not yet been clearly elucidated. In the present study, it was demonstrated that reversine of 5 μM could induce multinucleated cells through cytokinesis failure rather than just arrested in G2 or M phase. Moreover, reversine reversed the differentiation of sheep fibroblasts into MSC-like style, and notably increased the expression of pluripotent marker genes Oct4 and MSCs-related surface antigens. The fibroblasts treated with reversine could transdifferentiate into all three germ layers cells in vitro. Most importantly, the induced β-like cells and hepatocytes had similar metabolic functions with normal cells in vivo. In addition, reversine promoted fibroblasts autophagy, ROS accumulation, mitochondrial dysfunction and cell apoptosis via the mitochondria mediated intrinsic pathway. The results of high-throughput RNA sequencing showed that most differentially expressed genes (DEGs) involved in Mismatch repair, Nucleotide excision repair and Base excision repair were significantly up-regulated in reversine treated fibroblasts, which means that high concentration of reversine will cause DNA damage and activate the DNA repair mechanism. In summary, reversine can increase the plasticity of sheep fibroblasts and suppress cell growth via the mitochondria mediated intrinsic pathway.