N-Acetyl-l-methionine (NALM) is a Met derivative with lower cost, but it is unclear if it is effective as Met supplement for lactating dairy cows. The current study was conducted to investigate the effect of NALM supplementation on lactation performance and plasma variables in mid-lactating dairy cows. Forty-eight multiparous Holstein lactating cows with parity of 2.87 (±0.96 SD), and day in milk of 167.6 (±12.76 SD) were blocked into 12 groups and randomly assigned to one of 4 treatments: 0, 15, 30, or 60 g/d of NALM per cow to supplement the basal diet containing 2.0% Met (MP basis). The NALM was rumen protected, and supplementation of 10 g NALM is equivalent to 4.62 g Met. The experiment was conducted over a 13-week period, with the first week as adaptation. Milk production was recorded and milk samples were collected weekly; and dry matter intake was recorded biweekly. Blood samples were collected from the coccygeal vein at 3 h after the morning feeding on d 7 of wk 6 and wk 12. The data were analyzed using PROC MIXED of SAS, with covariance type AR (1) for repeated measures analysis. The yield of milk (P = 0.04), and milk lactose (P = 0.03) was increased quadratically, and energy-corrected milk yield (P = 0.06) tended to increase with increasing NALM supplementation in a quadratic manner. Dry matter intake, milk protein yield, milk fat yield, and contents of milk composition (protein, fat, lactose, milk urea nitrogen) were not affected by NALM supplementation. Plasma Met concentration (P = 0.04) increased quadratically, and proline (P = 0.08), total nonessential AA (P = 0.05) and total AA concentrations (P < 0.01) were significantly higher in 30 g/d group compared with those in the control group. Adding NALM tended to increase plasma concentrations of total protein (P = 0.06, quadratic) and globulin (P = 0.05, quadratic), but tended to decrease plasma urea nitrogen concentration (P = 0.08) in a quadratic manner. Meanwhile, plasma malonaldehyde concentration linearly decreased (P = 0.05) with increased doses of NALM. The results from this study demonstrate the positive effects of NALM on the lactation performance and oxidative stress of lactating dairy cows.