A study was carried out to determine the influence of nitrogen (N) sources on the growth, nitrate (NO3-) accumulation, and macronutrient concentrations of pakchoi (Brassica chinensis L.) in hydroponics. Plants were supplied with NO3- and two amino acids (AA), glutamic acid (Glu), and glutamine (Gln), at six NO3--N/AA-N molar ratios: (1) 100:0, (2) 80:20, (3) 60:40, (4) 40:60, (5) 20:80, (6) 0:100. The total N concentration was 12.5 mmol/L for all treatments in nutrient solutions. Both AAs reduced plant growth with decreasing NO3--N/AA-N ratios, but the reduction was for Gln than for Glu. At 80:20 NO3--N: Gln-N ratio, the Gln had no significant effect on pakchoi fresh weights. Decreasing NO3--N/AA-N ratios reduced NO3- concentrations in the plant, regardless of AA sources. Adding an appropriate portion of AA-N to nutrient solutions for hydroponic culture increased concentrations of N, phosphorus (P), and potassium (K) in pakchoi shoots. Substituting 20% or less of NO3--N with Gln-N in hydroponic culture will increase the pakchoi quality by reducing NO3- concentration and increasing mineral nutrient concentrations in shoots without significant reduction of crop yields.