Our objective was to determine the impact of feed restriction-induced negative energy balance on the fatty acid (FA) profile of lipid fractions in liver. Ten multiparous Holstein cows were randomly assigned to treatments of ad libitum feed intake (CON; n = 5) or feed restriction (RES; n = 5), with RES resulting in a mean energy balance of −13.1 ± 2.0 Mcal/d following 4 d of treatment. Cows were euthanized on d 4 and liver samples collected. Liver lipids were extracted, fractionated, and FA concentrations determined for triglycerides (TG), phospholipids (PL), cholesterol esters (CE), and free FA (FFA). The statistical model included the fixed effects of treatment, block, fraction, and interactions, as well as the random effect of cow nested within block. Compared with CON, RES altered the distribution of FA in liver lipid fractions. The proportion of FA in each fraction for CON and RES was 5.82% and 35.2% for TG (P < 0.01), 3.18% and 3.45% for FFA (P = 0.96), 88.1% and 59.0% for PL (P < 0.01), and 2.89% and 2.34% for CE (P = 0.91), respectively. Compared with CON, RES increased the concentration of C16:0 (P < 0.01 for all) and cis-9 18:1 (P < 0.01 for all) and decreased the concentration of C18:0 (P < 0.05 for all) in all fractions. The largest fold increase for C16:0 and cis-9 18:1 was in TG and FFA, respectively; the greatest fold decrease for C18:0 was in TG. The concentration (g/100 g FA) of C16:0, C18:0, and cis-9 18:1 for CON vs. RES was 27.2 vs. 40.7, 12.4 vs. 5.93, and 11.1 vs. 25.6 in TG, respectively; 16.1 vs. 21.8, 33.2 vs. 22.8, and 5.24 vs. 17.6 in FFA; 9.17 vs. 13.2, 31.6 vs. 28.2, and 7.09 vs. 14.8 in PL; and 15.6 vs. 21.8, 12.7 vs. 10.6, and 5.76 vs. 13.8 in CE. Compared with CON, RES decreased cis-9, cis-12 C18:2 (g/100 g FA) in TG (8.27 vs. 4.75; P < 0.01) but did not alter it in FFA (8.80 vs. 8.07; P = 0.36), PL (10.2 vs. 11.7; P = 0.35), or CE (25.7 vs. 22.6; P = 0.16). RES decreased cis-5, cis-8, cis-11, cis-14 C20:4 in TG (P < 0.01), FFA (P < 0.01), and CE (P = 0.08), but not in PL (P = 0.74). In conclusion, feed restriction induced-negative energy balance increased the proportion of FA in TG and increased C16:0 and cis-9 C18:1 in all liver lipid fractions.