In order to evaluate the effect of high-concentrate diet supplementation on milk protein content, six Holstein dairy cows were assigned into high-concentrate diet (HC) or low-concentrate diet (LC) groups (N = 3/group) for 50 days. With regard to milk protein, HC feeding significantly reduced the percentage of milk protein (P < 0.01), and milk protein yield also reduced. The milk somatic cell count numbers and N-acetyl-D-glucosaminidase activity was significantly higher (P < 0.01) in the HC group than in the LC group. A pre-column derivatization procedure of o-phthalaldehyde was used to analyze the milk amino acid profile, the contents of Asp, Gln, Ala, Ile, Leu, and Lys were significantly lower in milk (P < 0.05), but Arg and Phe were significantly higher (P < 0.05) in the HC group than in the LC group. The mRNA abundance for amino acid transporters SLC7A8, SLC7A10 (P < 0.05), SLC1A3 (P < 0.05), and SLC16A10 (P < 0.05) were decreased in the HC group. These data indicate that expression of amino acid transporters alters regulation of amino acid utilization and decreases milk quality in dairy cows.