Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host–pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2–dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.