Clinical trials testing the effects of a single injection of adenovirus carrying the human hepatocyte growth factor gene (Ad-HGF) in patients with chronic ischemic heart failure failed to show consistent improvements in cardiac function. The aim of this study was to evaluate the efficacy of repeated injections of Ad-HGF in a rat model of postinfarct heart failure. Ad-HGF or Ad-green fluorescent protein (GFP) was administered to Sprague Dawley rat models of postinfarct heart failure via single or fractional repeated intrapericardial injection. Heart function was monitored by magnetic resonance imaging for 4 and 8 weeks after injections. The expression of HGF or factor VIII/Ki-67 was evaluated by Western blot assay or immunofluorescence. We found that Ad-HGF gene expression could be prolonged in vivo by repeated injections and that cardiac function was significantly improved in the Ad-HGF repeat-injection group compared with the Ad-HGF single-injection group. Newly formed capillary density was similarly higher in the Ad-HGF repeat-injection group compared with that in the Ad-HGF single-injection group. We therefore conclude that fractional repeated injections of Ad-HGF may represent a promising therapeutic strategy to improve cardiac function in the setting of postinfarct heart failure.