Significance This work used mass spectrometry imaging to visualize a key bacterial virulence factor, lipid A within tissues infected by Francisella novicida , a model organism for Francisella tularensis ssp. tularensis , the causative agent of tularemia. Lipid components of the host were mapped using the same technique, leading to identification of a lethal role for host lipid metabolism in Francisella infection. Combined with profiling of lipid inflammatory mediators, this work defined the dramatic polarity of the cyclooxygenase-2 axis in this infection and overproduction of the proinflammatory product prostaglandin E2. The host–pathogen lipid interface was reconstructed to demonstrate the spatial organization of the host lipid response in the spleen.