The activity of NADPH oxidase (NOX) is blocked by nitric oxide (NO). Hydrogen sulfide (H<sub>2</sub>S) is also produced by blood vessels. It is reasonable to suggest that H<sub>2</sub>S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O<sub>2</sub><sup>–</sup> formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac<sub>1</sub> activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A<sub>2</sub> analogue U46619 ± NaHS for 1 or 16 h, and O<sub>2</sub><sup>–</sup> formation, NOX-1 expression and Rac<sub>1</sub> activity were assessed. The possible interaction between H<sub>2</sub>S and NO was also studied by using an NO synthase inhibitor, L-NAME, and an NO donor, DETA-NONOate. The role of K<sub>ATP</sub> channels was studied by using glibenclamide. NaHS inhibited O<sub>2</sub><sup>–</sup> formation following incubation of 1 h (IC<sub>50</sub>, 30 n M) and 16 h (IC<sub>50</sub>, 20 n M), blocked NOX-1 expression and inhibited Rac<sub>1</sub> activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H<sub>2</sub>S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac<sub>1</sub> and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H<sub>2</sub>S.