Objective— Hydrogen peroxide (H ₂ O ₂ ) is an important mediator in the vasculature, but its role in the regulation of soluble guanylate cyclase (sGC) activity and expression is not completely understood. The aim of this study was to test the effect of H ₂ O ₂ on sGC expression and function and to explore the molecular mechanism involved. Methods and Results— H ₂ O ₂ increased sGCβ1 protein steady-state levels in rat aorta and aortic smooth muscle cells (RASMCs) in a time- and dose-dependent manner, and this effect was blocked by catalase. sGCα2 expression increased along with β1 subunit, whereas α1 subunit remained unchanged. Vascular relaxation to an NO donor (sodium nitroprusside) was enhanced by H ₂ O ₂ , and it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly isolated vessels and RASMCs exposed to H ₂ O ₂ was greatly increased after sodium nitroprusside treatment. The H ₂ O ₂ -dependent sGCβ1 upregulation was attributable to sGCβ1 mRNA stabilization, conditioned by the translocation of the mRNA-binding protein HuR from the nucleus to the cytosol, and the increased mRNA binding of HuR to the sGCβ1 3′ untranslated region. HuR silencing reversed the effects of H ₂ O ₂ on sGCβ1 levels and cGMP synthesis. Conclusion— Our results identify H ₂ O ₂ as an endogenous mediator contributing to the regulation of vascular tone and point to a key role of HuR in sGCβ1 mRNA stabilization.