Long-term feeding of high-grain diets to dairy cows often results in systemic inflammation characterized by alterations in acute-phase proteins and other biomarkers, both in plasma and immune-responsive tissues like the liver. The systemic changes that underlie an acute high-grain feeding challenge remain unclear. The current study involved 6 Holstein and 6 Jersey cows in a replicated 3 × 3 Latin square. Periods (10 d) were divided into 4 stages (S): S1, d 1 to 3, served as baseline with TMR ad libitum; S2, d 4, served as restricted feeding, with cows offered 50% of the TMR fed on S1; S3, d 5, a grain challenge was performed, in which cows were fed a TMR ad libitum without (CON) or with an additional 20% pellet wheat-barley (1:1; HIG) top-dressed onto the TMR based on DMI obtained in S1; S4, d 6 to 10, served as recovery during which cows were allowed ad libitum access to the TMR. Among 28 biomarkers detected in plasma after 12 h of feeding on d 5, the concentrations of fatty acids and bilirubin increased in HIG Holstein but not in Jersey cows. In Holsteins, feeding HIG also increased total protein and albumin and decreased ceruloplasmin, myeloperoxidase and alkaline phosphatase concentrations. At the molecular level, however, hepatic genes associated with inflammation (IL1B, IL6, TNF, TLR4, MYD88, and NFKB1) were upregulated in Holsteins fed HIG compared with CON. Despite such response, expression of the acute-phase proteins SAA and HP in both Holsteins and Jerseys fed HIG compared with CON was markedly downregulated. In Holsteins fed HIG versus CON, the marked downregulation of SCD, ELOVL6, and MTTP together with upregulated CPT1A, ACOX1 and APOA5 indicated alterations in lipid metabolism during grain challenge. Genes related to TCA cycle and ketogenesis (PDK4, HMGCS2 and ACAT1) also were upregulated in Jerseys fed HIG. Data indicate that Jerseys tolerated better the high-grain challenge. An acute high-grain challenge in Holsteins induced marked alterations in lipid metabolism in the liver at least in part due to a localized inflammatory response. It remains to be determined if acute high-grain challenges can elicit long-term liver tissue damage, which could negatively affect the welfare of the cow.