Studies with isolated membrane fractions have shown that calmodulin (CaM) inhibits the activity of cardiac muscle cell Ca²⁺ release channel ryanodine receptor 2 (RyR2). To determine the physiological importance of CaM regulation of RyR2, we generated a mouse with 3 amino acid substitutions (RyR2-W3587A/L3591D/F3603A) in exon 75 of the Ryr2 gene, which encodes the CaM-binding site of RyR2. Homozygous mutant mice showed an increased ratio of heart weight to body weight, greatly reduced fractional shortening of the left ventricle, and lethality at 9–16 days of age. Biochemical analysis of hearts of 7- and 10-day-old homozygous mutant mice indicated an impaired CaM inhibition of RyR2 at micromolar Ca²⁺ concentrations, reduction in RyR2 protein levels and sarcoplasmic reticulum Ca²⁺ sequestration, and upregulation of genes and/or proteins associated with class II histone deacetylase/myocyte enhancer factor-2 and calcineurin signaling pathways. Sustained Ca²⁺ transients, often displaying repeated periods of incomplete Ca²⁺ removal, were observed in homozygous cardiomyocytes. Taken together, the data indicate that impaired CaM inhibition of RyR2, associated with defective sarcoplasmic reticulum Ca²⁺ release and altered gene expression, leads to cardiac hypertrophy and early death.