A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A 3 is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A 3 in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A 3, including phospholipase A 2 and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A 3 precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A 3 are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A 3 and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A 3 serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A 3 are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A 3 and 12-hydroxyeicosa-5Z, 8Z, 10E, 14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A 3 is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.