We have previously shown that increased nitric oxide (NO) production in sepsis impairs arteriolar conducted vasoconstriction cGMP-independently, and that the gap junction protein connexin37 (Cx37) is required for this conducted response. In the present study we hypothesized that NO impairs inter-endothelial electrical coupling in sepsis by targeting Cx37. We examined the effect of exogenous NO on coupling in monolayers of cultured microvascular endothelial cells derived from the hindlimb skeletal muscle of wild-type (WT), Cx37 null, Cx40 null and Cx43^G60S (nonfunctional mutant) mice. To assess coupling, we measured the spread of electrical current injected into the monolayer and calculated the monolayer intercellular resistance (inverse measure of coupling). The NO donor DETA rapidly and reversibly reduced coupling in cells from WT mice, cGMP-independently. NO scavenger HbO₂ did not affect baseline coupling, but it eliminated DETA-induced reduction in coupling. Reduced coupling in response to DETA was also seen in cells from Cx40 null and Cx43^G60S mice, but not in cells from Cx37 null mice. DETA did not alter the expression of Cx37, Cx40 and Cx43 in WT cells analyzed by immunoblotting and immunofluorescence. Furthermore, neither the peroxynitrite scavenger FeTPPS, superoxide scavenger MnTBAP, nor preloading of WT cells with the antioxidant ascorbate affected this reduction. We conclude that NO-induced reduction of electrical coupling between microvascular endothelial cells depends on Cx37, and propose that NO in sepsis impairs arteriolar conducted vasoconstriction by targeting Cx37 within the arteriolar wall.