Mesenchymal stem cells (MSCs) transplantation has emerged as a promising therapeutic strategy for acute myocardial infarction. However, there are still limitations for this therapy, such as low survival rate and poor cardiac differentiation potential of MSCs. In this study, we genetically engineered MSCs using ex vivo adeno-associated virus (AAV) transduction to overexpress Akt1 and Wnt11, which are well-characterized genes involved in MSC proliferation and cardiac differentiation. Our results showed that infection with AAV-Akt1-Wnt11 significantly upregulated the growth and proliferation of MSCs, as compared with those infected with AAV-Akt1 or AAV-Wnt11. In addition, co-expression of Akt1 and Wnt11 markedly promoted the expression of cardiac markers including NK2 transcription factor related 5, GATA-binding protein 4, α-myosin MHC and brain natriuretic protein. Notably, co-expression of Akt1 and Wnt11 increased cell survival and reduced cell apoptosis of MSCs under hypoxia/reoxygenation (H/R) treatment; however, these effects were blocked by Wnt11 neutralizing antibodies or Akt1 inhibitor. Moreover, co-culture of cardiomyocytes with MSCs infected with AAV-Akt1-Wnt11, in a dual chamber system, significantly reduced H/R-induced cell apoptosis compared with those co-cultured with MSCs infected with AAV-Akt1 or AAV-Wnt11. Overall, our results showed that MSCs, co-expressing Akt1 and Wnt11, showed greater survival and cardiac differentiation under H/R conditions and effectively ameliorated H/R-induced cardiomyocyte apoptosis in vitro. Our study suggests that transplantation of MSCs genetically engineered with AAV-Akt1-Wnt11 is a promising therapeutic strategy for treatment of acute myocardial infarction.