Robo4 maintains the barrier function of the mature vascular network by inhibiting endothelial permeability induced by pro-angiogenic factor and the function of Robo4 was tissue specific. The aim of this study was to investigate the role and signaling pathways of Robo4 in pulmonary endothelial permeability in Lipopolysaccharides (LPS) induced acute lung injury (ALI) in mice. Mice were challenged by intra-peritoneal (IP) injection of LPS (15 mg/kg) 48 h after gene transfer of Retro-hRobo4. Protein in bronchoalveolar lavage fluid (BALF), lung wet/dry ratio, lung injury score, MPO activity, secondary cytokine and survival rate was recorded. Co-immunoprecipitation (Co-ip) was used to detect the binding of Robo4 and RhoA. Robo4 mRNA and protein concentration in mice lungs were significantly increased after retrovirus-mediated gene delivery. Up-regulation of Robo4 significantly decreased protein in BALF, lung wet/dry ratio, lung injury score and MPO activity while intercellular tight junction protein ZO-1 was significantly increased. However, up-regulation of Robo4 didn’t change the concentration of TNF-a, vWF and VEGF in serum of mice. In addition, administration of Retro-hRobo4 significantly improved the survival of mice. Robo4 was bind to RhoA confirmed by Co-ip. These findings suggest that up-regulation of Robo4 enhanced pulmonary micro-vascular integrity and reduced lung edema in LPS induced ALI partially through binding to RhoA.