Sugarcane is a strategic roughage source for milk production in the tropics, and its supplementation with plant oils offers great potential for obtaining milk enriched with beneficial fatty acids, such as oleic (cis-9 C18:1), vaccenic (trans-11 C18:1), and rumenic (cis-9, trans-11 CLA) acids. The aim of this study was to evaluate the milk fatty acid composition of cows fed 60% chopped sugarcane-based diets containing citrus pulp and 0.0% (control), 1.5%, 3.0% and 4.5% sunflower oil on a dry matter basis. Twelve multiparous Holstein x Gyr dairy cows with an average milk production of 17±5 kg day-1 and 96±25 days in milk were allocated in a triplicate 4 x 4 Latin square design. There was no effect of dietary treatment on milk production, but sunflower oil supplementation linearly reduced the milk fat content and yield. The milk fat contents of lauric (C12:0), myristic (C14:0), and palmitic (C16:0) fatty acids were linearly reduced with increasing sunflower oil levels. There was a quadratic effect on the milk fat oleic, vaccenic, and rumenic acid contents, while the concentrations of elaidic acid (trans-9 C18:1), trans-10 C18:1 and trans-10, cis-12 CLA linearly increased in response to sunflower oil inclusion. Diet supplementation with sunflower oil linearly reduced both the atherogenicity and thrombogenicity indices and linearly increased the ratio between hypo- and hypercholesterolemic fatty acids in milk fat. The inclusion of up to 4.5% sunflower oil in 60% chopped sugarcane-based diets improved the nutritional quality of milk fat from Holstein x Gyr dairy cows as a result of the increased content of oleic, rumenic and vaccenic acids, which are beneficial to human health, and the concomitant reduction of hypercholesterolemic lauric, myristic and palmitic acids. However, there was an increase in the milk content of trans-10 C18:1 and elaidic acid, which are associated with deleterious effects on cardiovascular health.