Four multiparous lactating cows (175 to 220 d in milk [DIM]) were used in a 4×4 Latin square design to assess the effects of four doses (0.0, 0.5, 1.0, and 1.5μg/kg of body weight) of lipopolysaccharide (LPS; Escherichia coli 0111:B4) on performance and plasma metabolite and hormone concentrations. In addition, effects of immune activation on in vitro hepatic metabolic capacity were evaluated in 12 multiparous lactating cows (150 to 220 DIM) infused with 0 (n = 6), 1.0 (n = 4) or 2.0 (n = 2) μg of LPS/kg. Milk production and DMI decreased linearly with LPS dose for 24h after LPS infusion. Overall mean plasma tumor necrosis factor-α, insulin, glucagon, and cortisol concentrations increased linearly with LPS dose, and plasma β-hydroxybutyrate decreased linearly by dose after LPS infusion. Infusion of LPS decreased the insulin:glucagon molar ratio, but did not affect plasma concentrations of growth hormone, insulin-like growth factor-1, leptin, or l-(+)-lactate. Plasma concentrations of glucose tended to increase initially and subsequently decrease, and there was a quadratic tendency for increased plasma nonesterified fatty acid concentrations after LPS administration. In vitro hepatic capacity for conversion of [1-14C]l-(+)-lactate and [1-14C]palmitate, but not [1-14C]propionate or [1-14C]l-alanine, to CO2 increased after LPS administration. Hepatic capacity to convert [1-14C]propionate to glucose tended to increase, but neither esterification nor the conversion of palmitate to acid soluble products was altered by LPS. The LPS infusion resulted in significant changes of endocrine mediators responsible for regulation of energy metabolism of lactating cows and tended to alter subsequent in vitro hepatic metabolic capacity.