Abstract The effect of the content of lysine and methionine in metabolizable protein (MP) on lactation performance and N utilization in Chinese Holstein cows was determined. A control diet (C) was formulated to be adequate in energy but slightly limiting in MP. The concentration of Met and Lys in MP was 1.87 and 5.93%, respectively. The treatments were as follows (% of Met or Lys in MP): L=diet C supplemented with l-lysine-HCl at 0.49% on a dry matter (DM) basis (Met, 1.87; Lys, 7.00); M=diet C supplemented with 2-hydroxy-4-(methylthio)-butanoic acid (HMB) at 0.15% (Met, 2.35; Lys, 5.93); ML=diet C supplemented with 0.49% l-lysine HCl and 0.15% HMB (Met, 2.39; Lys, 7.10). The diets were fed to 60 Chinese Holsteins in mid-lactation (average days in milk=120, and milk yield=32.0 kg/d) for 8 wk. Milk yield was increased by supplementation of either Lys (1.5 kg/d) or Met (2.0 kg/d), and supplementation of both Lys and Met further increased milk yield (3.8 kg/d). There was no significant difference in dry matter intake across treatment groups. Cows on treatments M (3.95%) and ML (3.90%) had higher milk fat content than those on C (3.60%) and L (3.67%), but there were no significant differences in milk protein and lactose contents or somatic cell count among treatments. Supplementation of Met or Lys significantly increased Met or Lys concentration in arterial plasma. Treatment ML had a higher conversion of intake N to milk N and lower urea N concentrations in serum, urine, and milk than did treatment C. Supplementing HMB and l-lysine-HCl to provide approximately 2.3% Met and 7.0% Lys of the MP in diets slightly limiting in MP increased milk production, milk protein yield, and N utilization efficiency.