The re-expression of the embryonic transcription factor T-box 18 (TBX18) converts cardiomyocytes into pacemaker cells by somatic reprogramming. We tested whether adenoviral TBX18 gene transfer could create biological pacemaker activity in vivo in a large-animal model of complete heart block. This would prove whether this could be a viable therapeutic avenue for pacemaker-dependent patients afflicted with device-related complications. Biological pacemaker activity, originating from the intramyocardial injection site, was evident in TBX18-transduced animals. TBX18-transduced animals exhibited enhanced autonomic responses and physiologically superior chronotropic support of physical activity, compared to the controls. Induced sinoatrial node cells could be identified at the site of injection in TBX18-transduced animals. We further demonstrated that TBX18-induced biological pacemakers could restore antegrade conduction and improve cardiac dysfunction in pacemaker-induced cardiomyopathy. Therefore, in a clinically relevant disease model, the minimally invasive TBX18 gene transfer creates physiologically relevant pacemaker activity in complete heart block and reduces cardiac dysfunction in the pacemaker-induced cardiomyopathy.