The aim was to evaluate the impact of partially replacing a TMR with fresh alfalfa in lactating cows on the milk’s fatty acid profile (FA). Twelve multiparous cows were used in a change-over design and randomly assigned to one of two feeding strategies: T1) 100% TMR; T2) 60% TMR + fresh alfalfa grazed without limit of quantity. Each experimental period lasted 26 days (21 days adaptation, 5 days sampling). Fat was extracted from individual milk samples, and FA profile was determined by gas chromatography. Data were analyzed by ProcMixed and means separated by LSMeans (SAS®). The model included the effect of the period and the sequence of treatment application. Differences between periods were observed for some FA, which could be due to some changes in the diet composition during the study, a common problem in this kind of design. On the other hand, no differences were found in the sequence of treatment. Even if the UFA:SFA ratio was not improved, important changes in the milk FA profile were observed. Stearic acid (18:0) decreased in T2, which can provide health benefits for humans. The levels of α-Linolenic (18:3 c9, c12, c15), Rumenic (CLA, 18:2 c9, t11), and Vaccenic acid (TVA, 18:1 t11) were significantly higher on T2 (p<0.05), consistent with the use of pasture. The α-Linolenic in humans reduces the risk of chronic health issues, and Rumenic acid has been shown to bring strong physiological functions, especially anticarcinogenic effects.