Exocytosis of neutrophil granules contributes to acute lung injury (ALI) induced by infection or inflammation, suggesting that inhibition of neutrophil exocytosis in vivo could be a viable therapeutic strategy. This study was conducted to determine the effect of a cell-permeable fusion protein that inhibits neutrophil exocytosis (TAT-SNAP-23) on ALI using an immune complex deposition model in rats. The effect of inhibition of neutrophil exocytosis by intravenous administration of TAT-SNAP-23 on ALI was assessed by albumin leakage, neutrophil infiltration, lung histology, and proteomic analysis of bronchoalveolar lavage fluid (BALF). Administration of TAT-SNAP-23, but not TAT-control, significantly reduced albumin leakage, total protein levels in the BALF, and intra-alveolar edema and hemorrhage. Evidence that TAT-SNAP-23 inhibits neutrophil exocytosis included a reduction in plasma membrane CD18 expression by BALF neutrophils and a decrease in neutrophil granule proteins in BALF. Similar degree of neutrophil accumulation in the lungs and/or BALF suggests that TAT-SNAP-23 did not alter vascular endothelial cell function. Proteomic analysis of BALF revealed that components of the complement and coagulation pathways were significantly reduced in BALF from TAT-SNAP-23-treated animals. Our results indicate that administration of a TAT-fusion protein that inhibits neutrophil exocytosis reduces in vivo ALI. Targeting neutrophil exocytosis is a potential therapeutic strategy to ameliorate ALI.