I n heart failure (HF), Ca²⁺/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 Ca²⁺ 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.