Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) δ is the predominant cardiac isoform, and the δ_C splice variant is cytoplasmic. We overexpressed CaMKIIδ_C in mouse heart and observed dilated heart failure and altered myocyte Ca²⁺ regulation in 3-month-old CaMKIIδ_C transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca²⁺]_i transient amplitude, and diastolic [Ca²⁺]_i were all reduced by ≈50% in TG versus WT. This is explained by >50% reduction in SR Ca²⁺ content in TG versus WT. Peak Ca²⁺ current (I_Ca) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca²⁺ load and diastolic [Ca²⁺]_i, fractional SR Ca²⁺ release was increased and resting spontaneous SR Ca²⁺ release events (Ca²⁺ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca²⁺ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca²⁺ spark frequency and I_Ca, consistent with direct CaMKII activation of ryanodine receptors (and I_Ca) in TG. The rate of [Ca²⁺]_i decline during caffeine exposure was faster in TG, indicating enhanced Na⁺-Ca²⁺ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca²⁺ leak (via sparks), reduced SR Ca²⁺-ATPase expression, and increased Na⁺-Ca²⁺ exchanger explain the reduced diastolic [Ca²⁺]_i and SR Ca²⁺ content in TG. We conclude that CaMKIIδ_C overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.