Our previous study demonstrated that connexin (Cx)43 participates in the regulation of vascular permeability in severe sepsis. Whether OPN is involved in Cx43 regulation of vascular permeability after sepsis and the relationship with tight-junction proteins are not clear. In this study, we investigated the role of zo-1 and clauidn-5 in Cx43 regulation of vascular permeability and its relationship to OPN. The expression of zo-1 and claudin-5 were decreased in CLP rats and LPS-treated pulmonary vein VECs. Cx43 over-expressed lentivirus induced the degradation of zo-1 and claudin-5, while Cx43 RNAi lentivirus abrogate the degradation of zo-1 and cludin-5 induced by LPS. The vascular permeability and expression of OPN were significantly increased in CLP rats and LPS-treated pulmonary vein VECs. Silencing OPN by OPN RNAi lentivirus inhibited vascular hyper-permeability induced by LPS. Over-expressed Cx43 lentivirus increased the expression of OPN and vascular permeability, and down-regulated the expressions of zo-1 and claudin-5 in pulmonary vein VECs. Silencing OPN inhibited the effects of Cx43 over-expressed lentivirus on down-regulation of zo-1 and claudin-5 and vascular hyper-permeability in pulmonary vein VECs. Transfection of specific double-stranded RNA targeting to β-catenin and T-cell factor-4 (Tcf-4) abolished the up-regulation of OPN induced by Cx43 over-expression. These results suggest that OPN participates in the regulation of vascular permeability by Cx43 after sepsis. Cx43 up-regulation of OPN is via Tcf-4/β-catenin transcription pathway, OPN increases vascular permeability by decreasing the expressions of the tight junction proteins zo-1 and claudin-5.