A feeding trial was performed to determine the effects of rumen-protected lysine supplementation in lactating cattle diets differing in CP content. Twelve multiparous mid-lactation Holstein cows were blocked by DIM into a triplicate 4 × 4 Latin Square. Diets were arranged in 2 × 2 factorial design with 2 levels of CP (LO = 15%, HI = 17%) with or without lysine supplementation (+L, -L). Periods consisted of 14 d of adaptation followed by a sampling period of 3 d. During sampling periods, daily milk yield and DMI were recorded, and total collections of urine and feces were taken. Milk, urine, fecal, and plasma samples were collected for additional analysis. No significant interactions were found between CP content and Lys supplementation (P ≥ 0.14). DMI was affected by dietary CP (P ≤ 0.01) but not Lys supplementation (P = 0.21; LO-L = 25.3 kg/d; LO+L = 26.0; Hi-Ly = 27.5; Hi+L = 28.2; SEM = 1.42). Milk yield increased with CP content (P = 0.03) and Lys supplementation (P ≤ 0.01; LO-L = 34.6 kg/d; LO+L = 36.6; Hi-L = 36.1; Hi+L = 38.0; SEM = 1.87). Energy-corrected milk yields increased with dietary CP (P = 0.02) and supplemental Lys (P ≤ 0.01; LO-L = 34.98; LO+L = 37.06; Hi-Lys = 36.71; Hi+L = 39.02; SEM = 1.79). Milk fat (LO-L = 1.44 kg/d; LO+L = 1.52; Hi-Lys = 1.49; Hi+L = 1.60; SEM = 0.08) and milk protein (LO-L = 1.09 kg/d; LO+L = 1.18; Hi-Lys = 1.19; Hi+L = 1.25; SEM = 0.056) yields were raised by dietary CP (P = 0.05 and P ≤ 0.01 for fat and protein, respectively) and supplemental Lys (P = 0.01 for both fat and protein). Increased CP content increased milk protein %, milk urea nitrogen, and fecal N output (P ≤ 0.01), but there was no effect of Lys (P ≥ 0.56). No changes were seen in plasma glucose, NEFA, or BHBA as a result of either dietary CP or Lys supplementation (P > 0.10). The analysis shows that supplementing Lys in low CP diets can result in similar milk production to high CP diets without Lys supplementation.