The objective of this study was to evaluate the effects of antimicrobial virginiamycin on milk production and composition in high-producing dairy cows. In a commercial herd in Castro, Paraná State, Southern Brazil, 180 multiparous Holstein cows, bST-treated and milked 3x/day, were blocked by parity, milk yield and DIM. Within each block, cows were randomly allocated to 2 groups; control (CT) and treatment (VM), with VM group being supplemented with 300 mg/cow/day of virginiamycin (V-Max, Phibro Animal Health), and CT group receiving the same amount of corn meal. In the 5-d covariable period, cows had on average 2.9 ± 1.0 lactations, 111 ± 59 DIM, and 51.2 ± 6.4 kg/d milk yield. The experimental period had 6 weeks, and 3 milk collection periods were performed, totaling 45 milk samples for each cow. The corn silage and ryegrass haylage based-diet had on average 17.8% CP, 37.8% NDF, 17.7% ADF, 5.0% fat, and 7.4% ash. Milk composition (fat, protein, casein, lactose and total solids contents), as well as MUN and SCC were determined. Data were analyzed using the MIXED procedure of SAS with a model containing the effects of block, cow within treatment, period (wk 3 and 6), day, and treatment. Milk yield and its composition in the covariable period were included in the model as covariate. Virginiamycin supplemented cows produce more milk (50.34 vs. 49.31 ± 0.34 kg/d; P = 0.03), with higher milk protein (2.98 vs. 2.93 ± 0.01%; P < 0.01) and milk casein (2.32 vs. 2.27 ± 0.01%; P < 0.01) contents than control cows. Virginiamycin treated cows also show higher milk fat (1.704 vs. 1.640 ± 0.020 kg/d; P = 0.02), milk protein (1.471 vs. 1.424 ± 0.012 kg/d; P < 0.01), milk casein (1.143 vs. 1.106 ± 0.009 kg/d; P < 0.01), milk lactose (2.341 vs. 2.288 ± 0.017 kg/d; P = 0.03), and milk total solids (5.897 vs. 5.753 ± 0.051 kg/d; P < 0.05) amounts. No differences (P > 0.05) were found for milk fat, lactose, and total solids contents, as well as MUN and SCC linear score between controls and VM-treated cows. This work demonstrated virginiamycin improves milk production and milk composition, and it could become an important additive for lactating dairy cows.