During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17β-estradiol (InsHPrlE). The relative expression of β-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of β-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.