The optimization of milk production includes a rational use of forages, respect for the environment and offers the best quality to consumers. Milk production based on grass and forages produceshealthier milk and it is widely spread throughout the Atlantic arc to maximize milk yield per hectare. However, the mode of offeringthe grass can have a major influence on milk composition. The aim of this study was to evaluate the effect of grass supply mode (grazing, zero-grazing or ensiling) on dairy cows'performance, with particular reference to fatty acids and fat-soluble antioxidants concentration. A three by three Latin square experiment was performed with 18 dairy cows. Experimental treatments consisted of exclusive feeding with grass silageand zero-grazing, both offered ad libitum indoors, or grazing for 24 h. The results showed that grazing cows had a higher dry matter intake and greater milk yield than cows feeding on grass silage and zero-grazing, as well as higher concentrations of protein, lactose, nonfat-solidsand urea in milk than housed cows. Milk fatfrom grazing cows had a higher proportion of unsaturated fatty acids than from cows feeding on grass silage and zero-grazing, with significant differences in the proportion of vaccenic and rumenic acids. The 18:1 trans-11to 18:1 trans-10 ratio is proposed as biomarker to identify the milk produced from the management system of grazing cattle. Milk from grazing cows had a greater proportion of lutein than cows eating grass silage, with the zero-grazing system having intermediate values. In conclusion, the mode of grass supply affectsfatty acid and antioxidant profiles of milk.