Stimulation of the β-adrenergic system is important in the pathological response to sustained cardiac stress, forming the rationale for the use of β-blockers in heart failure. The β3-adrenoreceptor (AR) is thought to couple to the inhibitory G-protein, Gi, with downstream signaling through nitric oxide, although its role in the heart remains controversial. In this study, we tested whether lack of β3-AR influences the myocardial response to pressure-overload. Baseline echocardiography in mice lacking β3-AR (β3-/-) compared to wild type (WT) showed mild LV hypertrophy at 8 weeks that worsened as they aged. β3-/- mice had much greater mortality after transverse aortic constriction (TAC) than WT controls. By 3 weeks of TAC, systolic function was worse. After 9 weeks of TAC, β3-/- mice also had greater LV dilation, myocyte hypertrophy and enhanced fibrosis. NOS activity declined in β3-/-TAC hearts after 9 weeks, and total and NOS-dependent superoxide rose, indicating heightened oxidative stress and NOS uncoupling. The level of eNOS phosphorylation in β3-/-TAC hearts was diminished, and nNOS and iNOS expression levels were increased. GTP cyclohydrolase-1 expression was reduced, although total BH4 levels were not depleted. 3 weeks of BH4 treatment rescued β3-/- mice from worsened remodeling after TAC, and lowered NOS-dependent superoxide. Thus, lack of β3-AR signaling exacerbates cardiac pressure-overload induced remodeling and enhances NOS uncoupling and consequent oxidant stress, all of which can be rescued with exogenous BH4. These data suggest a cardioprotective role for the β3-AR in modulating oxidative stress and adverse remodeling in the failing heart.