Mutations in human cardiac calsequestrin (CASQ2), a high-capacity calcium-binding protein located in the sarcoplasmic reticulum (SR), have recently been linked to effort-induced ventricular arrhythmia and sudden death (catecholaminergic polymorphic ventricular tachycardia). However, the precise mechanisms through which these mutations affect SR function and lead to arrhythmia are presently unknown. In this study, we explored the effect of adenoviral-directed expression of a canine CASQ2 protein carrying the catecholaminergic polymorphic ventricular tachycardia–linked mutation D307H (CASQ2^D307H) on Ca²⁺ signaling in adult rat myocytes. Total CASQ2 protein levels were consistently elevated ≈4-fold in cells infected with adenoviruses expressing either wild-type CASQ2 (CASQ2^WT) or CASQ2^D307H. Expression of CASQ2^D307H reduced the Ca²⁺ storing capacity of the SR. In addition, the amplitude, duration, and rise time of macroscopic I_Ca-induced Ca²⁺ transients and of spontaneous Ca²⁺ sparks were reduced significantly in myocytes expressing CASQ2^D307H. Myocytes expressing CASQ2^D307H also displayed drastic disturbances of rhythmic oscillations in [Ca²⁺]_i and membrane potential, with signs of delayed afterdepolarizations when undergoing periodic pacing and exposed to isoproterenol. Importantly, normal rhythmic activity was restored by loading the SR with the low-affinity Ca²⁺ buffer, citrate. Our data suggest that the arrhythmogenic CASQ2^D307H mutation impairs SR Ca²⁺ storing and release functions and destabilizes the Ca²⁺-induced Ca²⁺ release mechanism by reducing the effective Ca²⁺ buffering inside the SR and/or by altering the responsiveness of the Ca²⁺ release channel complex to luminal Ca²⁺. These results establish at the cellular level the pathological link between CASQ2 mutations and the predisposition to adrenergically mediated arrhythmias observed in patients carrying CASQ2 defects.