Background: Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remain elusive. Previous work has shown that administration of a phospholipase A₂ (PLA₂) inhibitor attenuated postshock ALI and also identified a non-ionic lipid within the postshock mesenteric lymph (PSML) responsible for neutrophil (PMN) priming. Consequently, we hypothesized that gut-derived leukotriene B₄ (LTB₄) is a key mediator in the pathogenesis of ALI. Methods: Trauma/hemorrhagic shock (T/HS) was induced in male Sprague-Dawley rats and the mesenteric duct cannulated for lymph collection/diversion. PSML, arachidonic acid (AA) and a LTB₄ receptor antagonist were added to PMNs in vitro. LC/MS/MS was employed to identify bioactive lipids in PSML and the lungs. Results: T/HS increased AA in PSML and increased LTB₄ and PMNs in the lung. Lymph diversion decreased lung LTB₄ by 75% and PMNs 40%. PSML stimulated PMN priming (11.56±1.25 vs. 3.95±0.29 nmol O₂^-/min; /3.75x10⁵ cells/mL; p<0.01) that was attenuated by LTB₄ receptor blockade (2.64±0.58; p<0.01). AA stimulated PMNs to produce LTB₄, and AA-induced PMN priming was attenuated by LTB₄ receptor antagonism. Conclusion: Collectively, these data indicate that splanchnic I/R activates gut PLA₂-mediated release of AA into the lymph where it is delivered to the lungs provoking LTB₄ production and subsequent PMN-mediated lung injury.