Four fistulated Holstein cows were arranged in a 4 x 4 Latin square design to study the effects of graded amounts of glucose (0, 500, 750, and 1500 g/d) infused in the duodenum on milk yield and composition and plasma metabolites. Cows were fed a basal diet of 50% corn silage, 17% dehydrated alfalfa, and 33% concentrate. The treatments (feed plus infusions) were isoenergetic. Increased amounts of glucose did not affect milk yield or protein content. Fat yield and content decreased in a curvilinear manner; the lowest fat content was obtained with about 750 g of glucose. The decrease in milk fat resulted from a reduced yield of long-chain fatty acids (C16 and C18), probably caused by lower mobilization of fat. The glucose treatments significantly affected profiles of medium-chain fatty acids, which promoted the elongation process. The most important change in the plasma concentration of amino acids concerned decreased branched-chain amino acids. The lactose content was not greatly affected by infusions of glucose despite a significant linear increase in the concentration of milk glucose. In conclusion, an increase in the supply of glucose had no effect on milk yield and had a slight, positive effect on protein yield but induced a dramatic decrease in fat yield.