The level of milk production is closely linked to food availability in most mammals, including rodents. Previous studies have observed that changes in milk production parallel changes in blood insulin concentration, leading to speculation that insulin may be the primary regulator of nutritionally induced adjustments in mammary metabolism. In this study, mammary acini were isolated from normally fed and 18 h food-deprived lactating rats. Acini were treated with insulin and 2-D gel electrophoresis was used to compare the effects of food availability and insulin on the expression of 864 intracellular proteins (n=6). The expression of 26 proteins was found to be altered by insulin but only 7 were altered by overnight food deprivation. Proteins identified include enzymes involved in glycoprotein folding, lipid synthesis, mitochondrial metabolism, and cell protection. The regulation of most of these proteins has not previously been linked to food intake or insulin administration. The disparity in the effects of food availability and insulin on protein expression and the greater number of proteins determined to be affected by insulin suggests that they do not share the same mechanisms to regulate mammary metabolism.