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Interaction between host and pathogen determines the progression of an infection, thus understanding the metabolic context of this interaction is important for the treatment of pathogen-specific infections. However, because similar nutritional substrates and metabolic products are shared between host and pathogen, understanding their metabolic crosstalk is largely limited. For example, while amino acid metabolism supports host defensive responses against pathogens, the pathogens modulate amino acid metabolism for its survival and virulence. The co-incubation of Burkholderia thailandensis E264, a surrogate for cystic fibrosis (CF) pathogen B. pseudomallei, with murine lung epithelial cells revealed a metabolic crosstalk between the pathogen and epithelial cells. An LC-MS/MS-based untargeted metabolomics analysis yielded discriminative metabolites that delineate the crosstalk between the host cells and the pathogen. Here, we show the utility of pathway analysis to unravel differential metabolic pathways, underscoring the interaction between host and pathogen. Our results revealed that while aspartate metabolism has the highest impact, sphingolipid metabolism comprises the most dysregulated intermediates or metabolites, thus highlighting potential targets for the treatment of CF.