Altered Ca²⁺ homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via S-nitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S-nitrosylation, leading to altered Ca²⁺ homeostasis. Diastolic Ca²⁺ levels are elevated in NOS1^−/− and NOS1/NOS3^−/− but not NOS3^−/− myocytes compared with wild-type (WT), suggesting diastolic Ca²⁺ leakage. Measured leak was increased in NOS1^−/− and NOS1/NOS3^−/− but not in NOS3^−/− myocytes compared with WT. Importantly, NOS1^−/− and NOS1/NOS3^−/− myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1^−/− hearts, RyR2 S-nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca²⁺ leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease.