The objectives of this research were to develop a [U-¹³C]glucose tracer approach establishing the pathways and substrates for milk lactose and casein synthesis, and determine the influence of protein intake on murine mammary gland metabolism. Milk samples were collected after one, three and five days of feeding tracer (as 10% of dextrose). ¹³C-Isotopic and isotopomer plateaus were attained by day three, establishing the time-course necessary for tracer feeding. 23% of lactose-derived glucose originated from sources other than blood glucose. Six paired (intake and pups equal) sets of lactating mice were fed either a normal (20%) or low (10%) protein diet. ¹³C-mass isotopomer distribution (MID) in lactose-derived glucose and galactose did not differ, indicating common mammary metabolic pools. ¹³C-MID in blood versus casein indicated significant mammary synthesis of glutamate (Normal:51%, Low:50%), alanine (Normal:32%, Low:29%), and serine (Normal:18%, Low:37%, P < 0.05), suggesting additional requirements for glucose and/or EAA for NEAA synthesis.