Altered sphingolipid metabolism is associated with increased inflammation; however, the impact of inflammatory mediators, including neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. Using a well-characterized mouse model of NE oropharyngeal aspiration, we investigated a potential link between NE-induced airway inflammation and increased synthesis of various classes of sphingolipids, including ceramide species. Sphingolipids in bronchoalveolar lavage fluids (BAL) were identified and quantified using reverse phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis. BAL total and differential cell counts, CXCL1/keratinocyte chemoattractant (KC) protein levels, and high mobility group box 1 (HMGB1) protein levels were determined. NE exposure increased BAL long chain ceramides, total cell and neutrophil counts, and upregulated KC and HMGB1. The mRNA and protein levels of serine palmitoyltransferase (SPT) long chain subunits 1 and 2, the multimeric enzyme responsible for the first, rate-limiting step of de novo ceramide generation, were determined by Q/RT-PCR and western analyses, respectively. NE increased lung SPT long chain subunit 2 (SPTLC2) protein levels but not SPTLC1, and had no effect on mRNA for either subunit. To assess whether de novo ceramide synthesis was required for NE-induced inflammation, myriocin, a SPT inhibitor, or a vehicle control was administered intraperitoneally 2h prior to NE administration. Myriocin decreased BAL d18:1/22:0 and d18:1/24:1 ceramide, KC and HMGB1 induced by NE exposure. These results support a feed-forward cycle of NE-generated ceramide and ceramide-driven cytokine signaling that may be a potential target for intervention in lung disease typified by chronic neutrophilic inflammation.