Objective: To investigate the protection effect of over-expressed MicroRNA-22 on ischemic cardiomyocyte. Methods: Cardiomyocytes cultured in hypoxia and ischemia condition were transfected with recombinant adenovirus vector containing microRNA-22 plus a recombinant green fluorescent protein (GFP). Transduction efficiency and expression of microRNA-22 were assessed. The expression of downstream PTEN protein expression was detected. After successful transduction, cell activities including viability, nuclei proliferation and cell apoptosis were assessed. We used CCK-8 assay to assess cell activities, EdU assay to assess nuclei proliferation and Caspase-3/LDH assay to assess cell apoptosis. Results: MicroRNA-22 was transfected successfully using adeno-virus. The expression of microRNA-22 was significantly increased. The transduction efficiency reached more than 95%. Over-expression of microRNA-22 in cardiomyocytes down regulated PTEN protein level. Overexpression of microRNA-22 increased cell activities, DNA proliferation and decreased cell apoptosis. Conclusion: Over-expression of microRNA-22 protected cardiomyocytes from ischemic injury.