Enhancing the post-ruminal supply of arginine (Arg), a semi-essential amino acid (AA), elicits positive effects on milk production. Our objective was to determine the effects of Arg infusion on milk production parameters and aspects of nitrogen (N) absorption and utilization in lactating dairy cows. Six lactating Chinese Holstein cows of similar body weight (508 ± 14 kg), body condition score (3.0 ± 0), parity (4.0 ± 0), milk yield (30.6 ± 1.8 kg) and days in milk (20 ± 2 d) were randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 21 d for each period (1 week for infusion and 2 weeks for washout). Treatments were 1) Control: saline; 2) Arg group: saline + 9.42 g/L L-Arg; 3) Alanine (Ala) group: saline + 19.31 g/L L-Ala (iso-nitrogenous to the Arg group). Milk production and composition, dry matter intake, apparent absorption of N, profiles of amino acids (AA) in blood, urea N in urine, milk, and blood, and gene expression of AA transporters were determined. Compared with the Control or Ala group, the infusion of Arg led to greater expression of AA transporters (SLC7A2 and SLC7A8) and apparent uptake of free AA in the mammary gland, and was accompanied by greater milk yield, milk protein yield and milk efficiency (calculated by dividing milk yield over feed intake), together with lower concentration of urea N [regarded as an indicator of N utilization efficiency (NUE)] in blood and milk. Furthermore, in the cows infused with Arg, the NUE was higher and the concentration of urea N in urine was lower than those in the Ala group, although no differences were detected in NUE and urea N in urine between the Control and Arg group. The infusion of Ala had no effect on those indices compared with the Control. Overall, enhancing the post-ruminal supply of Arg via the jugular vein had a positive effect on the synthesis of milk protein at least in part by increasing gene expression of some AA transporters and uptake of free AA by mammary gland.