N-Carbamoylglutamate (NCG) is a structural analog of N-acetylglutamate, which is an allosteric activator of the essential auxiliary factor ammoniacal phosphate synthetase in the endogenous synthesis of Arg. In addition to its stability in the rumen, NCG is considered as a potential Arg enhancer for ruminants. This study was conducted to investigate the effect of NCG on milk production and composition in mid-lactating Holstein dairy cows. Sixty multiparous cows with a mean body weight of 669 kg (±71 SD) and day-in-milk of 176 (±55 SD) were blocked based on parity and milk production and randomly assigned into 4 treatments: basal diet supplemented with 0, 10, 20 or 40 g NCG per day per cow. The experiment was conducted according to a random block design with 14-wk periods, with the first 2 wk for an adaptation. Milk yield and composition were recorded weekly, while dry matter intake and plasma variables were determined every 2 wk. The data were analyzed using PROC MIXED of SAS. Addition of NCG had no effect on the dry matter intake and milk yield of the cows (P > 0.05). Milk fat content and yield increased linearly with NCG addition (P < 0.01). The contents of milk protein and total solid also increased linearly in the cows fed NCG (P < 0.05), whereas the yield of protein was not affected by the treatments (P > 0.05). Conversely, dietary addition of NCG increased the plasma nitric oxide content in a quadratic manner (P < 0.01). Moreover, addition of NCG linearly increased the plasma Arg content (P < 0.01). Overall, the results indicate that dietary NCG addition increased the milk protein and fat contents, which improved the milk quality of lactating dairy cows.