The objective of this study was to determine the effects of supplementation and withdrawal of a rumen-protected lysine product, AjiPro-L 2nd generation (2G), on production performance of high-producing dairy cows. Ninety-six multiparous Holstein cows averaging 2.9 ± 1.3 lactations and 83.6 ± 36.7 DIM at the commencement of the trial were assigned at random to one of 12 pens that received either a negative control ration (n = 4), positive control ration with added metabolizable lysine from an animal protein (n = 4), or AjiPro-L 2G added metabolizable lysine from a rumen-protected amino acid (n = 4). The 8wk experimental period consisted of 2 periods; a 4-wk treatment period followed by a 4-wk carryover period. During the treatment period, animals received negative, positive or AjiPro-L 2G treatments, whereas during the carryover period all animals received the positive control treatment. Milk yield, energy-corrected milk yield, water consumption and DMI were recorded daily and milk composition was measured weekly. Data from only cows that completed the entire duration of the study were used in the analysis. Days in milk categories of either high (114 ± 17.7) or low (53 ± 21.3) were also analyzed. Results showed that water consumption and DMI was significantly increased compared with baseline (P < 0.05) and MUN levels were significantly decreased compared with baseline (P < 0.01) for animals receiving AjiPro-L 2G during the treatment period. When comparing DIM categories, results revealed a significant difference (P < 0.05) in change of milk production, ECM, pounds of fat and pounds of lactose in comparison to baseline across treatments. For these parameters, high DIM animals receiving AjiPro-L 2G had greater milk yield, and the least reduction of ECM, pounds of fat, and pounds of lactose from baseline compared with the positive and negative treatments. During the carryover period all parameters measured, except for MUN (P < 0.05), were not different between treatments compared with baseline. Under the conditions of this experiment, supplementation of AjiPro-L 2G had the greatest effect on cows averaging 114 DIM in which milk production and milk components were maintained in post-peak cows.