Gap junctions (GJs) play an important role in vascular function, stability, and homeostasis in endothelial cells (ECs), and GJs are comprised of members of the connexin (Cx) family. GJs of vascular ECs are assembled from Cx37, Cx40, and Cx43, and we showed that ECs also express Cx32. In this study, we investigated a potential role for Cx32 during vascular inflammation. Expression of Cx32 mRNA and protein by human umbilical venous ECs (HUVECs) decreased following treatment with tumor necrosis factor (TNF)-alpha, but lipopolysaccharide (LPS) and interleukin (IL)-1beta did not affect Cx32 expression. Intracellular transfer of an inhibitory anti-Cx32 monoclonal antibody significantly enhanced TNF-alpha-induced monocyte chemotactic protein (MCP)-1 and IL-6 expression, but overexpression of Cx32 abrogated TNF-alpha-induced MCP-1 and IL-6 expression. LPS treatment of Cx32 knock-out mice significantly increased the serum concentrations of TNF-alpha, interferon-gamma, IL-6 and MCP-1, compared to wild-type littermate mice. These data suggest that Cx32 protects ECs from inflammation by regulating cytokine expression and plays an important role in the maintenance of vascular function.