BACKGROUND The permeability of pulmonary microvessel endothelial cells increases markedly after acute lung injury via paracellular gap. Connexin40 is a primary component of pulmonary microvessel endothelial cells gap junction channel and mediates intercellular communication. However, the relationship between connexin40 and the permeability of pulmonary microvessel endothelial cells is still unknown. Therefore, we determined whether connexin40 affected rabbits' pulmonary microvessel endothelial cells permeability after acute lung injury induced by gunshot trauma. METHODS We used an acute lung injury model in New Zealand rabbits following gunshot chest trauma and correlated connexin40 immunohistochemistry in gunshot lung tissue with Evans blue leak rate. Cultured pulmonary microvessel endothelial cells were divided into three groups, control (G control), injured serum (G serum), and blocker agent (G blocker). Gap junction channel function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer. RESULTS Connexin40 expression decreased time dependently, whereas Evans blue leak rate increased. Connexin40 expression and Evans blue leak rate exhibited a strong inverse correlation (gamma = -0.934, p < 0.05). Injured serum decreased gap junction channel function, and the gap junction channel blocker aggravated this effect. Similarly, pulmonary microvessel endothelial cells permeability increased significantly in G serum and G blocker. CONCLUSIONS Connexin 40 expression in pulmonary microvasculature endothelial cells is downregulated after acute lung injury induced by gunshot trauma. This is associated with impaired gap junction channel function and increased pulmonary microvessel endothelial cells permeability.