Objectives: The aim of this study was to determine whether activation of β3-adrenergic receptor (AR) and downstream signaling of nitric oxide synthase (NOS) isoforms protects the heart from failure and hypertrophy induced by pressure overload. Background: β3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike β1- and β2-ARs, β3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, serving as a "brake" to protect the heart from catecholamine overstimulation. Methods: C57BL/6J and neuronal NOS (nNOS) knockout mice were assigned to receive transverse aortic constriction (TAC), BRL37344 (β3 agonist, BRL 0.1 mg/kg/h), or both. Results: Three weeks of BRL treatment in wild-type mice attenuated left ventricular dilation and systolic dysfunction, and partially reduced cardiac hypertrophy induced by TAC. This effect was associated with increased nitric oxide production and superoxide suppression. TAC decreased endothelial NOS (eNOS) dimerization, indicating eNOS uncoupling, which was not reversed by BRL treatment. However, nNOS protein expression was up-regulated 2-fold by BRL, and the suppressive effect of BRL on superoxide generation was abrogated by acute nNOS inhibition. Furthermore, BRL cardioprotective effects were actually detrimental in nNOS -/- mice. Conclusions: These results are the first to show in vivo cardioprotective effects of β3-AR-specific agonism in pressure overload hypertrophy and heart failure, and support nNOS as the primary downstream NOS isoform in maintaining NO and reactive oxygen species balance in the failing heart.