Background Stimulation of soluble guanylate cyclase (sGC) or inhibition of phosphodiesterase type-5 (PDE5) activates protein kinase G-1α (PKG1α) to counteract cardiac hypertrophy and failure. PKG1α acts within localized intracellular domains; however, its oxidation at cysteine-42, linking homo-monomers, alters this localization, impairing suppression of pathological cardiac stress. Since PDE5 and sGC reside in separate micro-domains, we speculated that PKG1α oxidation might also differentially influence the effects from their pharmacological modulation. Methods and Results Knock-in mice expressing a redox-dead PKG1α (PKG1α C42S ) or littermate controls (PKG1α WT ) were subjected to trans-aortic constriction (TAC) to induce pressure-overload, and treated with a PDE5 inhibitor (sildenafil, SIL), sGC activator (BAY-602770, BAY), or vehicle. In PKG1α WT controls, SIL and BAY similarly enhanced PKG activity and reduced pathological hypertrophy/fibrosis and cardiac dysfunction after TAC. However, SIL failed to protect the heart in PKG1α C42S , unlike BAY, which activated PKG and thereby facilitated protective effects. This corresponded with minimal PDE5 activation in PKG1α C42S TAC versus higher activity in controls, and little colocalization of PDE5 with PKG1α C42S (versus co-localization with PKG1α WT ) in stressed myocytes. Conclusions In the stressed heart and myocytes, PKG1α C42 disulfide formation contributes to PDE5 activation. This augments the pathological role of PDE5 and so in turn enhances the therapeutic impact from its inhibition. PKG1α oxidation does not change the benefits from sGC activation. This finding favors the use of sGC activators regardless of PKG1α oxidation, and may help guide precision therapy leveraging the cGMP/PKG pathway to treat heart disease.