The dairy industry can benefit from the use of low crude protein (CP) diets due to improved N efficiency. However, His could become limiting in CP-deficient diets. Therefore, our objective was to evaluate the effect of rumen protected (RP)-His on production, and N and AA metabolism in dairy cows. Eight multiparous Holstein cows (130 ± 30 DIM, 42 ± 2 kg/d milk) were used in a replicated 4 × 4 Latin square design with 28-d experimental periods. Treatments included a basal diet composed (DM basis) of 50% corn silage, 15% haylage, and 35% concentrate supplemented with 0, 82, 164, and 246 g/d RP-His (Ajinomoto Inc.) and 11 g/d RP-Met (Smartamine M). Dietary His represented 2.06, 2.28, 2.52, and 2.75% of MP supply, respectively. Milk samples were collected in the last 2 wk of each period, with blood, urine (spot), and muscle samples in wk 4. Data were analyzed with the MIXED procedure of SAS, evaluating treatment effects with linear and quadratic contrasts. Treatments had no effect on DMI and milk composition. In contrast, yields of milk (30.8 to 32.9 kg/d; P = 0.01) and milk true protein (0.92 to 0.99 kg/d; P < 0.01) increased linearly, while milk fat yield (1.23 to 1.30 kg/d; P = 0.06) tended to increase linearly with RP-His. Plasma His (33.2 to 63.1 µM; P < 0.001) and carnosine (29.3 to 33.5 µM; P = 0.03), as well as muscle His (20.3 to 35.5 µM; P < 0.001) showed linear responses to RP-His, whereas no treatment effects were detected for plasma Met and Lys. There were no treatment effects on muscle carnosine and anserine. Milk urea-N (11.7 to 12.9 mg/dL; P = 0.03) and urinary excretion of urea-N (23.7 to 27.0% of N intake; P = 0.04) increased linearly in response to RP-His. Treatments did not affect urinary excretion of uric acid, allantoin, and total purine derivatives. Overall, supplementation with RP-His to a CP-deficient diet improved milk and milk protein synthesis, likely as a result of increased plasma His. Increased milk urea-N and urinary excretion of urea-N suggest that plasma His may have exceeded the requirement with excess deaminated and converted to urea in the liver due to moderate milk production in our study.