The spontaneous activity of pacemaker myocytes controls the heartbeat. Automaticity is due to the presence of the slow diastolic depolarization phase, which leads the membrane potential from the end of the repolarization phase to the threshold of the following action potential. f- (HCN) channels underlying the hyperpolarization-activated “funny” current (I_f) are thought to play a key role in the generation and autonomic regulation of the diastolic depolarization and heart rate, but their role is still subject of controversy. Here we show that conditional and time-controlled expression of a dominant-negative non-conductive human HCN4 channel subunit (hHCN4-AYA) in the mouse heart leads to virtually complete silencing of I_f current (>95%) in the diastolic depolarization range in the sino-atrial node and in the conduction system. Heart-specific I_f silencing induced sino-atrial bradycardia, sinus pauses, severe dysfunction of atrioventricular conduction and ventricular arrhythmias. In comparison to control myocytes, the basal automaticity of hHCN4-AYA SAN myocytes was reduced by 76% and by 67% in myocytes of the conduction system. However, the relative maximal positive chronotropic effect of badrenergic activation on in vivo heart rate, isolated atria or pacemaking of individual SAN and conduction myocytes was preserved showing that I_f does not play an exclusive role in heart rate regulation. Unexpectedly, crossing hHCN4-AYA mutant mice with mice lacking the cardiac muscarinic G-protein-activated channel Girk4 (Girk4^-/-) eliminated atrioventricular blocks and ventricular arrhythmias without preventing the autonomic regulation of heart rate. Our study shows, for the first time, the functional consequences of I_f silencing on heart rate and rhythm and indicates the possibility of managing cardiac disease related to HCN loss-of-function in humans by pharmacologic or genetic inhibition Girk4 channels.