In heart failure (HF), Ca 2+/calmodulin kinase II (CaMKII) expression is increased. Altered Na+ channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na+ channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIδ C on Na+ current (I Na) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIδ C overexpression shifted voltage dependence of Na+ channel availability by–6 mV (P< 0.05), and the shift was Ca 2+ dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2–related inhibitory peptide [AIP] or KN93). CaMKIIδ C markedly increased persistent (late) inward I Na and intracellular Na+ concentration (as measured by the Na+ indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIδ C overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na+ channels. In vivo, transgenic CaMKIIδ C overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na+ channels. This alters I Na gating to reduce availability at high heart rate, while enhancing late I Na (which could prolong action potential duration). In mice, enhanced CaMKIIδ C activity predisposed to VT. Thus, CaMKII-dependent regulation of Na+ channel function may contribute to arrhythmogenesis in HF.