Endotoxemia is a feature of steatohepatitis and sepsis. To define bovine metabolism in response to lipopolysaccharide (LPS) during a fatty acid (FA) insult, 10 multiparous Holstein mid-lactation cows were treated with a single i.v. bolus of saline (control; n = 5) or endotoxin (LPS E. coli O55:B5 at 0.375 μg/kg of BW; n = 5). Immediately post saline or LPS administration, all cows were i.v. infused a triglyceride (TG) emulsion (Intralipid 20% at 200 mL/h; Frasenius Kabi) for 16 h while fasted. Plasma was collected at h 0, 4, 8, 12, and 16, relative to bolus administration, start of TG infusion, and fasting. Liver was biopsied before (d −5) and after (h 16) these conditions. Plasma and liver metabolites were extracted for untargeted metabolomics or lipidomics using mass spec. Data were generalized log-transformed, auto-scaled, and analyzed using multivariate analyses. For metabolomics, metabolite identification was based on a mzCloud mass spectral score >80%. Mass spec detected 511 compounds. Although plasma FA (e.g., 18:2) levels increased with time (P < 0.05), only 50 of 122 hepatic TG (e.g., TG 16:0/18:2/18:2) increased by h 16 (P < 0.05). Whereas 25 of 122 hepatic TG decreased (P < 0.05). In liver, TG infusion with fasting increased 7 of 8 ceramides (P < 0.05). Plasma salicylic and 2-hydroxyhippuric acids, and leucine, tyrosine, and threonine levels decreased by h 16 of FA insult (P < 0.05). Pronounced reductions in plasma lysophosphatidylcholine (LPC) levels developed post LPS (17 of 18; e.g., LPC-16:0; P < 0.05). Similar reductions in plasma LPC:phosphatidylcholine ratios were observed (P < 0.05). Endotoxin increased plasma pyruvic and lactic acids, and microbial-derived phenylacetylglutamine (P < 0.05). Also, LPS decreased plasma citric and 5-aminovaleric acids, and leucine and ornithine levels (P < 0.05). In non-ruminants, LPC is an immune modulator and counteracts endotoxin-induced inflammation. Additionally, endotoxin promotes aerobic glycolysis and intestinal permeability, and inhibits intestinal leucine uptake. We conclude that LPS modifies the metabolome and lipidome of the lactating cow experiencing elevated circulating FA.