This study evaluates the intake and digestion of nutrients, parameters of rumen fermentation and degradation, omasal digesta and milk fatty acid composition, productive performance, and the concentration of serum metabolites in cows fed 600 g kg-1 sugarcane-based diets containing 0 (control), 15, 30, and 45 g kg-1 sunflower oil (SO) on a dry matter (DM) basis. Four rumen-cannulated Holstein x Gyr cows yielding 15±5 kg day-1 with 110±10 days in milk were allocated in a 4 x 4 Latin square design. Data were analyzed using mixed models, and significant differences were declared at P < 0.05. There was no effect of SO on the intake and apparent digestibility of DM, crude protein, neutral detergent fiber (NDF) and nonfibrous carbohydrates, but there was a linear increase in the intake and digestibility of ether extract. Dietary SO levels did not alter the ruminal degradability parameters for DM and NDF, rumen pH and contents of ammonia N, acetate, propionate and volatile fatty acids. Milk fat content and yield were linearly decreased, whereas a linear increase in milk protein content was observed in response to increasing levels of SO, but with no effect on milk yield. Linear reductions in palmitic and ?-linolenic acid contents, a linear increase in trans-10 C18:1 and elaidic acids, and a quadratic effect on vaccenic and rumenic acids were observed in omasal digesta of cows fed increasing levels of SO. Overall, up to 45 g kg-1 SO can be included on DM of chopped sugarcane-based diets without reducing consumption, apparent digestibility and rumen degradability of DM and fiber. Supplementing chopped sugarcane-based diets with 30 to 45 g kg-1 SO (DM basis) promotes milk fat depression due to the inhibition of mammary lipogenesis by specific rumen-derived fatty acid intermediates of the biohydrogenation of unsaturated C18 fatty acids. The inclusion of 15 to 45 g kg-1 SO in chopped sugarcane-based diets improves the nutritional quality of milk fat, with increases in the levels of oleic, vaccenic and rumenic acids, beneficial to human health, and a reduction in the levels of the hypercholesterolemic lauric, myristic and palmitic acids.