Absorption of glucose across the luminal membrane of absorptive enterocytes occurs via the Na⁺/glucose co-transporter-1 (SGLT1), while GLUT1 is the known major glucose transporter for glucose uptake by mammary acinar cells. AIMS: to compare i) SGLT1 expression and activity in intestinal tissues and ii) glucose transporter expression in the mammary gland of dry vs lactating cows. Three lactating and 3 dry Holstein cows were used. Multi-parous lactating cows were fed a TMR ad libitum and averaged (±SEM), 707 ± 26 kg BW, 21.8 ± 0.8 kg/d DMI, 356 ± 129 DIM, and 27.9 ± 9.9 kg/d milk yield. Dry cows were fed a grass silage and straw ration to meet maintenance requirements and averaged 688 ± 34 kg BW and 8.0 ± 0.4 kg/d DMI. Cows were euthanized and samples of duodenum, jejunum, ileum and mammary tissue, rapidly excised, were analyzed for mRNA, protein expression and glucose uptake using qPCR, immunohistochemistry, Western blotting, and U-¹⁴C-glucose uptake by brush boarder membrane vesicles (BBMV). Effects of tissue type and lactation were evaluated using ANOVA and Dunnett’s t-tests. In both lactating and dry cows intestinal SGLT1 mRNA, protein, and activity were highest in the duodenum and lowest in the ileum. In lactating vs dry cows, there was a 1.8- (P = 0.02), 3.8- (P = 0.02), and 2.8-fold (P = 0.01) increase in SGLT1 mRNA expression in the duodenum, jejunum, and ileum, respectively. This was matched by increased SGLT1 protein abundance in BBMV by 3.1- (P = 0.04), 5.6- (P = 0.01), and 5.1-fold (P = 0.01) in the duodenum, jejunum, and ileum, correlating well with 3.0- (P = 0.02), 6.8- (P = 0.03), and 5.9-fold (P = 0.02) increase in initial rates of Na⁺-dependent glucose transport into BBMV isolated from duodenum, jejunum, and ileum. We believe this is the first study showing SGLT1 protein being co-localized with GLUT1 on the basolateral membrane of bovine mammary gland acinar cells. There was a 3.6- (P = 0.05) and 9.3-fold (P = 0.01) increase in GLUT1 and SGLT1 expression in mammary tissue of lactating vs dry cows. Greater basolateral membrane expression of GLUT1 and SGLT1 in mammary secretory cells during lactation enhances glucose uptake and is accompanied by increased expression and activity of intestinal SGLT1.