The aim of this study was to evaluate the ruminal fermentation and degradation parameters, the kinetic flow of digesta in the gastrointestinal tract, and the milk fatty acid (FA) profile in Holstein x Gyr cows fed 59% sugarcane silage-based diets with 0% (control), 5%, 10% and 15% whole cottonseed (WCS) on a dry matter (DM) basis. Four rumen-cannulated cows with an average milk yield of 14.4±3.3 kg day-1 and 85±25 days in milk were allocated in a 4 x 4 Latin square design. There was no effect of dietary WCS levels on the intake of organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and nonfibrous carbohydrates, but there was a linear increase in the intake of ether extract (EE). There was no treatment effect on the apparent digestibility of DM, OM, CP, EE and NDF or on the ruminal degradability parameters for DM and NDF of sugarcane silage. Also, no treatment effects were observed on rumen pH, ruminal and postruminal particulate passage rates or on the rate of passage of the fluids in the rumen. WCS promoted linear reductions in the milk fat contents of lauric, myristic and palmitic acids and linear increases in the milk fat contents of stearic, oleic, trans-10 C18:1 and elaidic acids. The milk fat contents of vaccenic, rumenic, linoleic and ?-linolenic acids were unaffected by WCS supplementation. The inclusion of up to 15% WCS in sugarcane silage-based diets did not alter the digestibility of nutrients, the rumen degradability of the fiber, or the kinetic flow of digesta in the gastrointestinal tract of Holstein x Gyr cows but improved the nutritional quality of milk fat through an increase in the content of oleic acid, which is beneficial to human health, and decreased levels of hypercholesterolemic lauric, myristic and palmitic acids.