Signaling via endothelial nitric oxide synthase (NOS3) limits the heart’s response to -adrenergic (-AR) stimulation, which may be protective against arrhythmias. However, mechanistic data are limited. Therefore, we performed simultaneous measurements of action potential (AP-using patch clamp), Ca²⁺ transients (Fluo-4), and myocyte shortening (edge detection). L-type calcium current (I_Ca) was directly measured by the whole cell ruptured patch clamp technique. Myocytes were isolated from wildtype (WT) and NOS3 knockout (NOS3^-/-) mice. NOS3^-/- myocytes exhibited a larger incidence of -AR (isoproterenol- 1 µM)-induced early afterdepolarizations (EADs) and spontaneous activity (defined as aftercontractions). We also examined I_Ca, a major trigger for EADs. NOS3^-/- myocytes had a significantly larger -AR-stimulated increase in I_Ca compared to WT myocytes. In addition, NOS3^-/- myocytes had a larger response to -AR stimulation compared to WT myocytes in Ca²⁺ transient amplitude, shortening amplitude, and AP duration (APD). We observed similar effects with specific NOS3 inhibition (L-NIO, 10 µM) in WT myocytes as with NOS3 knockout. Specifically, L-NIO further increased isoproterenol-stimulated EADs and aftercontractions. L-NIO also further increased the isoproterenol-stimulated I_Ca, Ca²⁺ transient amplitude, shortening amplitude, and APD (all P<0.05 vs isoproterenol alone). L-NIO had no effect in NOS3^-/- myocytes. These results indicate that NOS3 signaling inhibits the -AR response by reducing I_Ca and protects against arrhythmias. This mechanism may play an important role in heart failure, where arrhythmias are increased and NOS3 expression is decreased.