Feeding dairy cows large proportions of cereal grain is commonly associated with rumen acidosis, activation of innate immunity, and perturbation of intermediary metabolism. We previously showed that steeping barley grain in 0.5% lactic acid (LA) decreased the rate of starch degradation, lowered the risk of subacute rumen acidosis, modulated rumen fermentation profile, and increased milk fat content in dairy cows. This study sought to investigate whether feeding of LA-treated barley grain would affect carbohydrate and lipid metabolism as well as innate immunity. Eight rumen-fistulated late-lactation (approximately 217 d in milk, DIM) Holstein cows were randomly assigned, in a 2 × 2 crossover design, to 1 of the 2 dietary treatments consisting of 27% (dry matter basis) rolled barley grain steeped for 48 h in an equal volume (wt/vol) of tap water (CTR) or 0.5% LA (TRT). Each experimental period lasted 21 d, with the first 11 d for diet adaptation. Blood and rumen samples were collected on d 12, 15, 17, and 21 of the experimental period before the morning feeding to evaluate the effects of dietary treatment on preprandial day-to-day variation of plasma and rumen variables. To establish the effect of treatment on diurnal variation of plasma variables, blood samples were collected on the last day of each period at 0, 2, 4, 6, 8, 10, and 12 h after the morning feeding (i.e., 0800 h). Results of the day-to-day study showed that cows fed the TRT diet had greater overall preprandial concentrations of glucose, cholesterol, and insulin, and a lower concentration of haptoglobin in plasma. Diurnal data indicated lower concentrations of haptoglobin and serum amyloid A and a tendency for greater plasma lactate in cows fed the TRT diet. A treatment by time interaction was observed for glucose, lactate, insulin, haptoglobin, and lipopolysaccharide-binding protein, suggesting a role for both the processing of grain and the time of sampling on those variables. No effect of diet on plasma concentrations of cortisol, β-hydroxybutyrate, and nonesterified fatty acids or rumen endotoxin was evidenced. Taken together, our results demonstrated that feeding barley grain steeped in 0.5% LA modulated both energy status and innate immunity of dairy cows fed relatively high levels (45% of dry matter) of dietary concentrate.