Background— Catecholaminergic polymorphic ventricular tachycardia is characterized by stress-triggered syncope and sudden death. Patients with catecholaminergic polymorphic ventricular tachycardia manifest sinoatrial node (SAN) dysfunction, the mechanisms of which remain unexplored. Methods and Results— We investigated SAN [Ca 2+ ] i handling in mice carrying the catecholaminergic polymorphic ventricular tachycardia–linked mutation of ryanodine receptor (RyR2 R4496C ) and their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2 R4496C mice after isoproterenol injection, analogous to what was observed in catecholaminergic polymorphic ventricular tachycardia patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca 2+ ] i transients in SAN cells within the intact SAN by confocal microscopy. RyR2 R4496C SAN presented significantly slower pacemaker activity and impaired chronotropic response under β-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca 2+ ] i transients and action potentials) in 75% of the cases. Ca 2+ spark frequency was increased by 2-fold in RyR2 R4496C SAN. Whole-cell patch-clamp experiments performed on isolated RyR2 R4496C SAN cells showed that L-type Ca 2+ current ( I Ca,L ) density was reduced by ≈50%, an effect blunted by internal Ca 2+ buffering. Isoproterenol dramatically increased the frequency of Ca 2+ sparks and waves by ≈5 and ≈10-fold, respectively. Interestingly, the sarcoplasmic reticulum Ca 2+ content was significantly reduced in RyR2 R4496C SAN cells in the presence of isoproterenol, which may contribute to stopping the “Ca 2+ clock” rhythm generation, originating SAN pauses. Conclusion— The increased activity of RyR2 R4496C in SAN leads to an unanticipated decrease in SAN automaticity by a Ca 2+ -dependent decrease of I Ca,L and sarcoplasmic reticulum Ca 2+ depletion during diastole, identifying subcellular pathophysiological alterations contributing to the SAN dysfunction in catecholaminergic polymorphic ventricular tachycardia patients.