The aim was to evaluate the impact of partially replacing a TMR with fresh alfalfa in lactating cows on the Danbo cheese’s fatty acid profile (FA). In a cross-over design experiment, twelve cows were randomly assigned to one of two feeding strategies: T1) 100% TMR and T2) 60% TMR + fresh alfalfa grazed without limit of quantity. Each experimental period lasted 26 days. After 21 days of adaptation, the milk of the morning milking was collected from each cow and a cheese/cow was made the same day (n=12 cheeses/period). After 90 days of maturation, the FA profile was determined by gas chromatography. Data were analyzed by ProcMixed and means separated by LSMeans (SAS®). There were differences between the periods, but the sequence of treatment application did not influence the study. Even if SFA:UFA ratio did not differ between groups, the content of Stearic acid (18:0) decreased in T2. This acid is one of the main SFA in dairy products, often associated with cardiovascular diseases in humans. The levels of Rumenic (CLA, 18:2 c9, t11), Vaccenic (TVA, 18:1 t11), and α-Linolenic acid (18:3 c9, c12, c15), increased in T2, all of them with health benefits. Moreover, total trans-FA was raised by almost 20% in T2. In summary, these findings indicate that partially replacing a TMR for fresh pasture in cow’s diet could be an option to increase the contribution of FA with beneficial repercussions in human health, such as anticarcinogenic effects provided especially by CLA.