The present study was designed to explore the role of soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway in sulfur dioxide (SO2)-induced vasodilation. We showed that SO2induced a concentration-dependent relaxation of phenylephrine (PE)-precontracted rat aortic rings in association with an increase in cGMP concentration, whereas L-aspartic acid β-hydroxamate (HDX), an inhibitor of SO2synthase, contracted rings in a dose-dependent manner. Pretreatment of aortic rings with the sGC inhibitor ODQ (30 μM) attenuated the vasodilatory effects of SO2, suggesting the involvement of cGMP pathway in SO2-induced vasodilation. Mechanistically, SO2upregulated the protein levels of sGC and PKG dimers, while HDX inhibited it, indicating SO2could promote cGMP synthesis through sGC activation. Furthermore, the dimerization of sGC and PKG and vasodilation induced by SO2in precontracted rings were significantly prevented by thiol reductants dithiothreitol (DTT). In addition, SO2reduced the activity of phosphodiesterase type 5 (PDE5), a cGMP-specific hydrolytic enzyme, implying that SO2elevated cGMP concentration by inhibiting its hydrolysis. Hence, SO2exerted its vasodilatory effects at least partly by promoting disulfide-dependent dimerization of sGC and PKG, resulting in an activated sGC/cGMP/PKG pathway in blood vessels. These findings revealed a new mode of action and mechanisms by which SO2regulated the vascular tone.