The dairy industry can benefit from low crude protein (CP) diets due to reduced N excretion, but shortages of Met, Lys, and His may limit milk protein synthesis. We studied the effect of incremental amounts of rumen-protected (RP)-His on plasma and muscle AA profile, nutrient utilization, and yields of milk and milk true protein in dairy cows. Eight multiparous Holstein cows (130 ± 30 d in milk) were randomly assigned to treatment sequences in a replicated 4 × 4 Latin square design with 28-d experimental periods. Treatments included a basal diet composed (dry matter basis) of 50% corn silage, 15% haylage, and 35% concentrate supplemented with 0, 82, 164, and 246 g/d of RP-His and 11 g/d of RP-Met. Milk, plasma, and muscle samples were collected weekly or every other week during all 4 periods, whereas spot urine and fecal grab samples were taken only in wk 4 of each period. Data were analyzed individually by week using linear, quadratic, and cubic orthogonal polynomials and repeated measures. Plasma His increased linearly with RP-His during wk 1 (30.3 to 57.2 µM) to wk 4 (33.2 to 63.1 µM). Plasma carnosine increased linearly with supplemental RP-His except in wk 2. No treatment effect was observed for plasma 3-methylhistidine except a quadratic effect in wk 3. Inclusion of RP-His showed linear effects on muscle His in wk 2 (20.1 to 32.5 µM) and 4 (20.3 to 35.5 µM). Whereas muscle anserine and carnosine concentrations were not affected by treatments in wk 4, anserine responded quadratically and carnosine showed a trend for a quadratic response to RP-His in wk 2. During wk 4, treatments did not affect urinary excretion of total purine derivatives, as well as dry matter intake and milk concentrations of fat and true protein. In contrast, milk yield tended to increase linearly (31.2 to 32.7 kg/d) and milk true protein yield responded linearly (0.93 to 0.98 kg/d) and tended to increase quadratically to RP-His supplementation in wk 4. Also, milk urea-N (11.7 to 12.9 mg/dL) and urinary excretion of urea-N (23.7 to 27.0% of N intake) increased linearly with feeding RP-His in wk 4. Overall, RP-His was effective to enhance plasma and muscle concentrations of His and milk protein synthesis. Elevated milk urea-N and urinary excretion of urea-N suggest that plasma His may have exceeded the requirement with excess N converted to urea in the liver. Future research is needed to determine the bioavailability of RP-His supplements to improve the accuracy of diet formulation for AA.