We established previously that 5-HT _2B receptors are involved in cardiac hypertrophy through the regulation of hypertrophic cytokines in cardiac fibroblasts. Moreover, the generation of reactive oxygen species and tumor necrosis factor-α through the activation of reduced nicotinamide-adenine dinucleotide phosphate [NAD(P)H] oxidase has been implicated in cardiac hypertrophy. In this study, we investigated whether 5-HT _2B receptors could be involved in the development of cardiac hypertrophy associated with superoxide anion production. Therefore, we measured the effects of serotonergic 5-HT _2B receptor blockade on left-ventricular superoxide anion generation in 2 established pharmacological models of cardiac hypertrophy, ie, angiotensin II and isoproterenol infusions in mice. Angiotensin II infusion for 14 days increased superoxide anion concentration (+32%), NAD(P)H oxidase maximal activity (+84%), and p47 ^phox NAD(P)H oxidase subunit expression in the left ventricle together with hypertension (+37 mm Hg) and cardiac hypertrophy (+17% for heart weight:body weight). The 5-HT _2B receptor blockade by a selective antagonist (SB215505) prevented the increase in cardiac superoxide generation and hypertrophy. Similarly, infusion for 5 days of isoproterenol increased left-ventricular NAD(P)H oxidase activity (+48%) and cardiac hypertrophy (+31%) that were prevented by the 5-HT _2B receptor blockade. Finally, in the primary culture of left-ventricular cardiac fibroblasts, angiotensin II and isoproterenol stimulated NAD(P)H oxidase activity. This activation was prevented by SB215505. These findings suggest that the 5-HT _2B receptor may represent a new target to reduce cardiac hypertrophy and oxidative stress. Its blockade affects both angiotensin II and β-adrenergic trophic responses without significant hemodynamic alteration.