BACKGROUND: β-AR (β-adrenergic receptor) stimulation regulates atrial electrophysiology and Ca ²⁺ homeostasis via cAMP-dependent mechanisms; however, enhanced β-AR signaling can promote atrial fibrillation (AF). CNP (C-type natriuretic peptide) can also regulate atrial electrophysiology through the activation of NPR-B (natriuretic peptide receptor B) and cGMP-dependent signaling. Nevertheless, the role of NPR-B in regulating atrial electrophysiology, Ca ²⁺ homeostasis, and atrial arrhythmogenesis is incompletely understood. METHODS: Studies were performed using atrial samples from human patients with AF or sinus rhythm and in wild-type and NPR-B-deficient (NPR-B ^+/− ) mice. Studies were conducted in anesthetized mice by intracardiac electrophysiology, in isolated mouse atrial preparations using high-resolution optical mapping, in isolated mouse and human atrial myocytes using patch-clamping and Ca ²⁺ imaging, and in mouse and human atrial tissues using molecular biology. RESULTS: Atrial NPR-B protein levels were reduced in patients with AF, and NPR-B ^+/− mice were more susceptible to AF. Atrial cGMP levels and PDE2 (phosphodiesterase 2) activity were reduced in NPR-B ^+/− mice leading to larger increases in atrial cAMP in the presence of the β-AR agonist isoproterenol. NPR-B ^+/− mice displayed larger increases in action potential duration and L-type Ca ²⁺ current in the presence of isoproterenol. This resulted in the occurrence of spontaneous sarcoplasmic reticulum Ca ²⁺ release events and delayed afterdepolarizations in NPR-B ^+/− atrial myocytes. Phosphorylation of the RyR2 (ryanodine receptor) and phospholamban was increased in NPR-B ^+/− atria in the presence of isoproterenol compared with the wildtypes. C-type natriuretic peptide inhibited isoproterenol-stimulated L-type Ca ²⁺ current through PDE2 in mouse and human atrial myocytes. CONCLUSIONS: NPR-B protects against AF by preventing enhanced atrial responses to β-adrenergic receptor agonists.