We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca(2+) ([Ca(2+)](i)) and Ca(2+) sensitivity to the myofilament. Here we show that G6PD is activated by membrane depolarization via PKC and PTEN pathway and that G6PD inhibition decreases intracellular free calcium ([Ca(2+)](i)) in vascular smooth muscle cells and thus arterial contractility. In bovine coronary artery (CA), KCl (30 mmol/l) increased PKC activity and doubled G6PD V(max) without affecting K(m). KCl-induced PKC and G6PD activation was inhibited by bisperoxo(pyridine-2-carboxyl)oxovanadate (Bpv; 10 μmol/l), a PTEN inhibitor, which also inhibited (P < 0.05) KCl-induced CA contraction. The G6PD blockers 6-aminonicotinamide (6AN; 1 mmol/l) and epiandrosterone (EPI; 100 μmol/l) inhibited KCl-induced increases in G6PD activity, [Ca(2+)](i), Ca(2+)-dependent myosin light chain (MLC) phosphorylation, and contraction. Relaxation of precontracted CA by 6AN and EPI was not blocked by calnoxin (10 μmol/l), a plasma membrane Ca(2+) ATPase inhibitor or by lowering extracellular Na(+), which inhibits the Na(+)/Ca(2+) exchanger (NCX), but cyclopiazonic acid (200 μmol/l), a sarcoplasmic reticulum Ca(2+) ATPase inhibitor, reduced (P < 0.05) 6AN- and EPI-induced relaxation. 6AN also attenuated phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Ser855, a site phosphorylated by Rho kinase, inhibition of which reduced (P < 0.05) KCl-induced CA contraction and 6AN-induced relaxation. By contrast, 6AN increased (P < 0.05) vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239, indicating that inhibition of G6PD increases PKA or PKG activity. Inhibition of PKG by RT-8-Br-PET-cGMPs (100 nmol/l) diminished 6AN-evoked VASP phosphorylation (P < 0.05), but RT-8-Br-PET-cGMPs increased 6AN-induced relaxation. These findings suggest G6PD inhibition relaxes CA by decreasing Ca(2+) influx, increasing Ca(2+) sequestration, and inhibiting Rho kinase but not by increasing Ca(2+) extrusion or activating PKG.