The liver plays a key role regulating fatty acid metabolism and gluconeogenesis and failure to adapt during lactation may lead to decreased milk yield and pathologies in dairy cows. Thus, our aim was to study the effect of pasture grazing on changes in liver composition during lactation. Multiparous Holstein cows (n = 24, 664 ± 65 kgBW, 3.0 ± 0.4 BCS, spring calving) were assigned in a randomized block design to (G0) a total mixed ration (TMR) fed ad libitum (70% forage: 30% concentrate) or (G1) grazing plus supplementation from 0 to 180 d postpartum (DPP). The G1 cows grazed Festuca arundinacea or Medicago sativa in 2 (18 h) or 1 (6 h) session depending on heat stress (30 or 20 kgDM/cow/day) and were supplemented with 5.4 kgDM of a commercial concentrate or offered TMR (50% of G0 offer). From 180 to 250 DPP, all cows grazed Festuca arundinacea (10h; 30 kgDM/cow/day) and were offered TMR (50% of G0 offer). Liver biopsies and blood samples were collected at −14, 35, 60, 100, 180 and 250 DPP and protein, free glucose, glycogen and triglyceride concentrations were determined. Data were analyzed with a mixed model using repeated measures. Liver protein peaked (P < 0.001) at 100 DPP for both treatments. While free liver glucose increased (P < 0.001) only at 35 DPP, hepatic glycogen decreased (P < 0.001) in early lactation (35 DPP), increased during mid-lactation and dropped again at 250 DPP. Hepatic glycogen was higher for G0 than G1 cows (1.66 vs. 1.31 ± 0.07% m/m, P < 0.01). Glycogen mobilization at 35 DPP could have led to increased free glucose since the correlation was negative (r = −0.4, P < 0.01). Liver triglyceride concentrations tended to be affected (P = 0.06) by the interaction between treatment and DPP increasing only at 35 DPP for G1 (10.2 vs. 6.3 ± 0.7%, G1 vs. G0 respectively, P < 0.05) and remaining unchanged during the rest of lactation for both treatments. Plasma insulin correlated positively with glycogen (r = 0.3, P < 0.01) and tended to correlate negatively with triglyceride (r = −0.2, P = 0.08), confirming the role of this hormone in lipid and glucose metabolism. Our results suggest that liver composition is modified during lactation and nutrition may affect energy reserves.