Connexin (Cx)43 has been shown to participate in several cardiovascular diseases. Increased vascular permeability is a common and severe complication in sepsis or septic shock. Whether or not Cx43 takes part in the regulation of vascular permeability in severe sepsis is not known and the underlying mechanism has not been described. Using cecal ligation and puncture-induced septic rats and lipopolysaccharide (LPS)-treated vascular endothelial cells (VECs) from pulmonary veins, the role of Cx43 in increased vascular permeability and the relationship to the Rho A/Rock1 pathway were studied. It was shown that vascular permeability in the lungs, kidneys, and mesentery in sepsis rats and LPS-stimulated monolayer pulmonary vein VECs were significantly increased and positively correlated with the increased expression of Cx43 and Rock1 in these organs and cultured pulmonary vein VECs. Connexin inhibitor, carbenoxolone(10mg/kg,iv) and Rock1 inhibitor,Y-27632(2mg/kg,iv) alleviated the vascular leakage of lung, mesentery and kidney in sepsis rats. Over-expressed Cx43 increased the phosphorylation of 20 k Da myosin light chain (MLC₂₀) and the expression of Rock1, and increased the vascular permeability and decreased the transendothelial electrical resistance of pulmonary vein VECs. Cx43 RNA interference decreased the phosphorylation of MLC₂₀ and the expression of Rock1, and decreased LPS-stimulated hyperpermeability of cultured pulmonary vein VECs. The Rock1 inhibitor, Y-27632, alleviated LPS- and over-expressed Cx43-induced hyperpermeability of monolayer pulmonary vein VECs. This report shows that Cx43 participates in the regulation of vascular permeability in sepsis and the he mechanism is related to the Rock1-MLC₂₀ phosphorylation pathway.