Eicosanoids are a group of bioactive lipids, shown to be important mediators of neutrophilic inflammation, and selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 (HxA3) is a pathogen-elicited, epithelial-produced neutrophil chemoattractant that directs trans-epithelial migration in response to infection. Following HxA3-driven trans-epithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2 (PLA2), and the cytosolic PLA2α (cPLA2α) isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil trans-epithelial migration has not been explored. Human and mouse neutrophil-epithelial co-cultures were employed to evaluate the role of neutrophil-derived cPLA2α in infection-induced trans-epithelial signaling using pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-Optical Coherence Tomography (μOCT), a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil trans-epithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil trans-epithelial migration by mediating leukotriene B4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.