Milk fat synthesis requires butyrate, acetate, and reducing equivalents (NADPH) as substrates. NADPH is provided from glucose through the pentose phosphate pathway and isocitrate dehydrogenase (IDH1). During milk fat depression (MFD), reduced milk fat synthesis may result in decreased uptake of nutrients by the mammary gland, including glucose. The objective of this study was to characterize expression of glucose metabolism and NADPH synthesis enzymes in mammary tissue during MFD using tissue from a previously published experiment. Cows were arranged in a 3 × 3 Latin square design with 14-d experimental periods. Treatments were control (CON), CLA-induced MFD (CLA; 3 d i.v infusion of 10 g/d of trans-10,cis-12 CLA in an intralipid emulsion), and a low forage, high oil diet (LF/HO; 45.9% forage, 3.0% soybean oil, and 1.5% fish oil). Milk fat yield was decreased 24% by CLA and 38% by LF/HO. Mammary biopsies were performed 1 to 3 h after milking at the end of each treatment. Gene expression of enzymes involved in glucose metabolism and NADPH synthesis were determined by Real-Time PCR and analyzed relative to the geometric mean of housekeeping genes (18S ribosomal subunit, ribosomal protein S9 and β2-microglobulin). Data were analyzed using the fit model procedure of JMP Pro and the model included fixed effects of treatment and geometric mean of housekeeping genes and random effects of cow and period. Glucose transporter 1 (GLUT1) and IDH1 were significantly reduced by the LF/HO treatment when compared with CON (28 and 36%, respectively; P < 0.05). Phosphogluconate dehydrogenase expression was lower in the LF/HO treatment when compared with CLA (P < 0.05), but not different compared with CON. Other genes involved in glucose metabolism such as HK2, ACLY, G6PDH and GLUT8 were not affected by treatments.These results suggest that during diet-induced MFD the reduction in glucose uptake coincides with decreased expression of GLUT1 and that the decrease in use of NADPH corresponds to a downregulation of the isocitrate pathway. The smaller decrease in milk fat in the CLA treatment may have limited the ability to observe a treatment effect during CLA-induced MFD.