Objectives were to evaluate the effects of Intralipid (IL; 20% i.v. fat emulsion; Fresenius Kabi, Uppsala, Sweeden) infusion on metabolism, inflammation, and liver lipid content following an i.v. lipopolysaccharide (LPS) challenge in lactating cows. Cows (765 ± 32 kg BW; 273 ± 35 DIM) were enrolled in 2 experimental periods (P); during P1 (5d) baseline data were obtained. At the start of P2 (2d), cows were assigned to 1 of 2 treatments: 1) control + IL (CONIL; 3 mL of sterile saline; n = 5) or 2) LPS + IL (LPSIL; 0.375 μg/kg of BW LPS; n = 5). Directly following i.v. bolus (saline or LPS) administration, IL was i.v. infused continuously (200 mL/h) for 16h. Cows were fasted for 16h during P2. Liver biopsies were obtained on d1 of P1 and at 16 and 48h postbolus. Effects of treatment, time, and treatment × time interactions were assessed using PROC MIXED (SAS Inst. Inc., Cary, NC). Mild pyrexia (0.8°C) was observed for 5.5h postbolus in LPSIL relative to CONIL cows (P = 0.01). LPS increased circulating insulin (4-fold) during the IL infusion period relative to CONIL cows (P < 0.01). Circulating glucagon increased 3-fold in LPSIL cows from 8 to 12h post-LPS relative to CONIL (P = 0.03). NEFA concentrations gradually increased in both treatments (3-fold, relative to baseline; P ≤ 0.04), but peaked (43%) higher in CONIL compared with LPSIL cows (P = 0.01). Circulating BHB decreased in both treatments for the first 8h of P2, after which it gradually increased. Infusing IL increased (36%) liver TG content in CONIL cows at 16 h relative to baseline (P = 0.05), but it had no effect on lipid content of LPSIL cows. No treatment differences in liver lipid content were observed at 48h. Relative to CONIL, circulating LPS-binding protein in LPSIL cows increased 2-fold at 8h postbolus then markedly decreased (5-fold; P < 0.01). Serum amyloid A concentrations progressively increased in LPSIL cows during P2 (3-fold, relative to CONIL; P < 0.01). In summary, IL infusion altered the characteristic patterns of insulin and LBP in response to LPS, but did not cause fatty liver.