The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN) have been identified as a key factor of cardiac pacemaker activity. The present study investigated the feasibility of using transfection of HCN4, an important subunit in the HCN family, to cure an experimental cardiac bradyarrhythmia. Two adenoviral vectors containing HCN4 and GFP (Ad-HCN4) or GFP alone (Ad-GFP) were constructed. Three or four days after gene injection, the pigs underwent catheter ablation of the atrioventricular (AV) node. After a complete AV block was created, the idioventricular heart rate in the Ad-HCN4 group was significantly greater than in controls. The heart rhythm in the Ad-HCN4 group could be modulated by the beta-adrenergic agonist isoproterenol. An I(f) current could be observed in the ventricular myocytes of the Ad-HCN4 group under patch clamp technique investigations. The expected cell membrane localization of GFP-tagged HCN4 expression was confirmed with confocal fluorescent microscopy. The successful in vivo transfection with Ad-HCN4 was also identified by real-time reverse transcription polymerase chain reaction (RT-PCR). Our study suggested that site-specific gene therapy for cardiac bradyarrhythmias using adenoviral vectors to overexpress HCN4 channels might be feasible.