Acute pancreatitis accompanied by a subsequent infectious attack can often lead to multisystem organ dysfunction, including acute lung injury (ALI), but the molecular mechanisms are poorly defined. In this study we explored the role of the priming insult by induction of cerulein pancreatitis, which was followed by the second attack due to endotoxemia, in the development of ALI in mice. Experiments revealed that lipopolysaccharide (LPS) injection in mice with acute pancreatitis caused the development of ALI as indicated by blood-gas derangements, pulmonary vascular hyperpermeability, increased inflammatory cell counts in broncho-alveolar lavage, and histological lung damage. This was associated with the pancreatitis-induced increase in expression of macrophage migration inhibitory factor (MIF) in lungs, together with elevated expression of Toll-like receptor (TLR)-4, both of which were inhibited by administration of anti-protease-activated receptor (PAR)-2 antibody. Furthermore, anti-MIF antibody treatment suppressed the pancreatitis-induced elevation of TLR-4 pulmonary expression. Genetic removal of MIF from mice resulted in less development of ALI in the setting of acute pancreatitis complicated by endotoxemia. These findings demonstrate that activation of PAR-2 with trypsin, which can be released after pancreatitis induction, positively regulates the transcript level of MIF and increased MIF results in exaggerated pulmonary expression of TLR-4, leading to the development of ALI with a subsequent infectious attack. We thus suggest that interventions designed to modulate MIF may have therapeutic advantage in treating ALI in patients with acute pancreatitis complicated by bacterial infection.