<div><p>The objective of this study was to evaluate the effect of whole cottonseed on diets of cows under grazing on fatty acid profile and milk cholesterol. Five crossbred Holstein × Zebu cows were distributed in a 5 × 5 Latin square. Inclusion levels were 0, 6, 12, 18 and 24%. There was a linear decrease in all fatty acids quantified, saturated and monounsaturated (p > 0.05), possibly due to the action of polyunsaturated fatty acids ingested in the diet. There was no influence of treatment on conjugated linoleic acids or cholesterol. It is concluded that, regardless of inclusion level, there is a modification in the milk lipid profile, reducing the concentration of saturated fatty acid, monounsaturated fatty acid, and making it rich in polyunsaturated fatty acids, which are beneficial to health.</p></div