Acute lung injury (ALI) is associated with high morbidity and mortality in critically ill patients. At present, the functional contribution of airway mucins to ALI is unknown. We hypothesized that excessive mucus production could be detrimental during lung injury. Initial transcriptional profiling of airway mucins revealed a selective and robust induction of MUC5AC upon cyclic mechanical stretch exposure of pulmonary epithelia (Calu-3). Additional studies confirmed time- and stretch-dose-dependent induction of MUC5AC transcript or protein during cyclic mechanical stretch exposure in vitro or during ventilator-induced lung injury in vivo. Patients suffering from ALI showed a 58-fold increase in MUC5AC protein in their bronchoalveolar lavage. Studies of the MUC5AC promoter implicated nuclear factor κB in Muc5ac induction during ALI. Moreover, mice with gene-targeted deletion of Muc5ac⁻/⁻ experience attenuated lung inflammation and pulmonary edema during injurious ventilation. We observed that neutrophil trafficking into the lungs of Muc5ac⁻/⁻ mice was selectively attenuated. This implicates that endogenous Muc5ac production enhances pulmonary neutrophil trafficking during lung injury. Together, these studies reveal a detrimental role for endogenous Muc5ac production during ALI and suggest pharmacological strategies to dampen mucin production in the treatment of lung injury.