Objective: To investigate the effect of cardiomyocyte proliferation induced by human hepatocyte growth factor (HGF) in pigs with chronic myocardial infarction (CMI). Methods: A steerable, deflectable 7F catheter incorporating a 27-guage needle was advanced percutaneously to the left ventricular myocardium of 18 pigs with CMI. Pigs were randomized (1:1:1) to receive adenoviral vector HGF (total dose, 1×1010 genome copies), which was administered as five injections into the infarcted myocardium (total, 1.0 mL), or saline, or Ad-null (control groups). Injections were guided by Ensite NavX left ventricular electroanatomical mapping. HGF and cyclin proteins were detected by western blot and immunoprecipitation analysis. Histological and immunohistochemical analysis determined proliferating cardiomyocytes. Myocardial perfusion and cardiac function were estimated by Gated-Single Photon Emission Computed Tomography (G-SPECT). Results: Western blot analyses showed that HGF were predominantly expressed in the infarct core and border in the myocardium of the infarcted heart. G-SPECT analysis indicated that the HGF group had better cardiac function and myocardial perfusion four weeks after the injection of Ad-HGF than before the injection of Ad-HGF. After treatment there were more proliferating cardiomyocytes in the HGF group compared to either of the control groups. Furthermore, the HGF group myocardial samples expressed higher levels of p-Akt, cyclin A, cyclin E, cyclin D1, cdk2, cdk4 than those in the control groups. Conclusion: The over-expression of HGF activates pro-survival pathways, induces cardiomyocyte proliferation, and improves the perfusion and function of the porcine CMI heart.