The effect of intestinal glucose supply on mammary utilization of amino acids (AA) was studied in four lactating dairy cows. Glucose (0, 443, 963, and 2398 g/d) was continuously infused in the duodenum over 14-d periods using a Latin square design. A grass silage-based diet was formulated so that treatments (diet + infusions) were isoenergetic and isonitrogenous and met 100 and 110% of energy and protein requirements, respectively. Mammary AA uptake was determined by arteriovenous difference and continuous blood flow measurement. The milk protein yield tended to be quadratically increased (to +88 g/d for 963 g of glucose) by glucose infusion, but milk protein content was not significantly affected. Treatments did not change significantly arterial concentrations of urea and glucogenic AA. Mammary arterial fluxes of essential AA increased linearly with glucose infusion, whereas fluxes of nonessential and glucogenic AA were not significantly affected. Mammary arteriovenous differences and extraction rates were roughly unchanged by treatments. Mammary uptake of all essential AA, excluding Arg and Val, increased linearly with increasing supply of glucose. Ratio of blood AA uptake to milk protein output increased significantly for His, Met, and Leu. For the highest infused dose of glucose, all AA except for His were taken up in excess relative to their secretion in milk. Based on evolution of extraction rate and ratio of uptake to output, His and Leu could have limited the milk protein yield response to glucose infusions.