The objective of this study was to determine the effects of rumen-protected Lys (RP-Lys) supplementation in dairy cows on feed intake, AA in blood, and milk production and milk composition. Thirty-six Holstein dairy cows, averaging 123 (±32) DIM, 653 (±67) kg body weight, and 38.3 (±5.0) kg/d of milk production were housed in individual tie stalls and fed one of 3 diets as a TMR. Treatments (TRT) were a control diet (CON) with no added RP-Lys, a diet containing 0.3% of RP-Lys (0.3RP-Lys), and a diet containing 0.6% of RP-Lys (0.6RP-Lys). Dry matter intake (DMI) and milk yield were measured daily and milk composition, BW and BCS were measured weekly. Data were analyzed using the MIXED procedure of SAS with the fixed effects of TRT and wk with random effects of cow within TRT. Means were compared using Tukey’s multiple comparison and were considered to be different when P < 0.05 and tended to differ when 0.05 ≤ P ≤ 0.1. There were no differences in DMI (26.2 ± 3.3 kg DM, P = 0.97), milk yield (37.8 ± 5.5 kg/d; P = 0.41) and milk composition between TRT (% Protein 3.02 ± 0.22, P = 0.30; % Fat 3.38 ± 0.69, P = 0.70). There was no effect of TRT on total essential AA (EAA) in plasma (916, 893, 862 ± 44.5 μM, for CON, 0.3RP-Lys, and 0.6RP-Lys, respectively). Plasma His was reduced in a linear fashion by Lys feeding (P < 0.05, with 45.68, 41.28, 32.80 ± 3.661 μM, for CON, 0.3RP-Lys and 0.6RP-Lys, respectively). Plasma Lys and Met, as a % of EAA, increased linearly (P < 0.05) with RP-Lys supplementation. There was a tendency (0.05 ≤ P ≤ 0.1) for cows fed 0.6RPLys to have higher plasma Lys as % EAA than cows fed CON (5.72, 5.94, and 6.46 ± 0.242% EAA for CON, 0.3RP-Lys and 0.6RP-Lys respectively). In conclusion, cows supplemented with 0.6RP-Lys had similar DM intake and milk production despite elevated plasma Lys. The data indicate post-ruminal delivery of RP lysine. The decrease in plasma His suggests the inability of the cows used in this study to respond with changes in milk production linked to AA limitations other than additional Lys. Support for VMRM was from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil(CAPES).