Activation of Ca2+-sensitive, large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) by local, ryanodine receptor-mediated Ca2+ signals (Ca2+ sparks) acts as a brake on pressure induced (myogenic) vasoconstriction—a fundamental mechanism that regulates blood flow in resistance arteries. Here, we report a novel mechanism linking physiological intraluminal pressure within small arteries to Ca2+ spark/BK channel vasodilation: oxidative activation of VSMC cGMP-dependent protein kinase (PKG) through formation of an oxidant-induced disulphide bond between cysteine residues (Cys42). Third-order mesenteric arteries were studied from knock-in mice expressing a PKG variant in which Cys42 is replaced with serine; the resulting PKG[C42S] variant is resistant to oxidant-induced activation but can still be activated normally by cGMP. PKG[C42S]KI arteries displayed significantly enhanced intraluminal pressure-induced constriction (80 mmHg) compared with WT arteries (WT: 32.4 ± 1.2% [n = 26] vs PKG[C42S]KI: 39.8% ± 2.5% [n = 21], P <0.05) and almost entirely absent BK vasodilation (1 µM Paxilline constriction; WT: 11.4 ± 1.8% [n = 6] vs PKG[C42S]KI: 1.3% ± 0.6% [n = 5], P <0.01). Furthermore, the non-specific PKG inhibitor DT-2 constricted WT arteries but had no effect on PKG[C42S]KI artery diameters (3 μM DT-2 constriction; WT: 10.9 ± 3.1% [n = 6] vs PKG[C42S]KI: 1.7 ± 1.3 [n = 4], P <0.01). Epifluorescent imaging of CM-H2DCFDA loaded arteries demonstrated pressure-induced oxidant production and Western blot protocols demonstrated both oxidant- and pressure-induced PKG dimerization in mesenteric arteries. Perforated patch clamp studies of mesenteric VSMCs revealed absence of spontaneous transient outward currents from PKG[C42S]KI VSMCs at -40mV (WT: 0.92 ± 0.57 Hz [n = 6] vs PKG[C42S]KI: 0.06 ± 0.04 Hz [n = 6], P <0.05) but conversely, whole cell voltage step protocols indicated equivalent BK channel I/V characteristics. High speed confocal microscopy of pressurised arteries loaded with the Ca2+ indicator Fluo-4 revealed significant reduction in Ca2+ sparks in PKG[C42S]KI arteries compared with WT (see figure). Importantly, exogenous H2O2 increased Ca2+ spark frequency in unpressurised WT arteries but not in PKG[C42S]KI arteries (see figure). Values reported are means ± SEM, compared by Student’s t-test. Our interpretation is that disablement of the oxidant activating mechanism in the PKG[C42S]KI mice reduces Ca2+ spark frequency, decreasing pressure-induced BK channel activation and thereby deactivating the BK channel-mediated negative feedback regulation of vasoconstriction. Therefore, our results support the novel concept of a negative feedback control mechanism that regulates arterial diameter through mechanosensitive production of oxidants to activate PKG and enhance Ca2+ sparks.