The objectives were to evaluate the effects of dietary fish oil on plasma metabolite, hepatic fatty acid composition, and total triacylglycerol concentrations. Multiparous Holstein cows (n=42) were completely randomized to 1 of 3 treatments at 3wk prepartum. Treatments were no supplemental lipid or supplemental lipid from either Energy Booster (Milk Specialties Co., Dundee, IL) or fish oil. Treatment diets were fed from −21d relative to expected date of parturition until 10d postpartum. Treatments were fed as a bolus before the a.m. feeding. The dose of lipid fed during the prepartum period was 250g, whereas approximately 0.92% of the previous day's dry matter intake was supplemented postpartum. Blood was collected 3 times weekly for determination of plasma metabolites. Liver biopsies were performed at 21 and 10d before expected date of parturition and 1 and 14d after parturition to determine fatty acid compositions and total triacylglycerol concentrations. Dry matter intake, milk yield, and loss of body weight or body condition score were not affected by supplementing the diet with lipid or by the source of lipid. Supplemental lipid tended to increase plasma glucose and decrease nonesterified fatty acids during the postpartum period. Furthermore, plasma β-hydroxybutyrate was reduced during the postpartum period in the lipid-supplemented treatments. However, source of supplemental lipid had no influence on any blood metabolite. Supplemental fish oil altered the fatty acid composition of liver phospholipids and triacylglycerols, decreasing total saturated fatty acids and increasing total n-3 and long-chain polyunsaturated fatty acids (>20 carbon fatty acids). Despite the altered fatty acid composition, hepatic total triacylglycerol concentrations were unaffected by supplemental fish oil. Furthermore, the improved metabolic profile following lipid supplementation did not decrease hepatic total triacylglycerol concentrations.