Glucose transport across the plasma membrane is suggested to be a rate-limiting step in milk synthesis. Glucose transporter 1 (GLUT1) is a predominant glucose transporter in the lactating bovine mammary glands. Branched-chain amino acids (BCAA) increased GLUT1 expression and the translocation to plasma membrane in muscle and intestinal tissues in rats and pigs. An abomasal infusion of casein however decreased the clearance of glucose by the mammary glands in cows infused with starch. The objective of this study was to examine the impact of BCAA on the abundance of GLUT1 in bovine mammary epithelial cells (BMEC). Primary BMEC from 4 passages (n = 4) were treated with media rich in all essential amino acids (+EAA), deficient in only BCAA (−BCAA), or deficient in all essential amino acids (−EAA) for 24 h. The amino acid concentrations in –BCAA and –EAA were 10% of +EAA. All media contained bovine insulin (100 μg/L) and d-glucose (17.5 mmol/L). Cell membrane and cytosolic protein fractions were extracted and 15 μg of protein from each fraction was subjected to Western immunoblotting analyses. The abundance of total GLUT1 (GLUT1-t) was calculated by adding together the abundance of GLUT1 in cytosolic (GLUT1-c) and cell membrane (GLUT1-m) protein fractions. The GLUT1-m was expressed as a fraction of GLUT1-t to describe the intensity of translocation of GLUT1 from cytoplasm to the cell membrane. Similarly, the abundance of β-actin in both protein fractions were determined. The ratio of β-actin in the membrane fraction to total β-actin was calculated and used to explain potential cytosolic protein contaminations in the membrane protein fraction. Treatment effects were analyzed using the MIXED procedure in SAS including treatment, corresponding β-actin value as a covariate and cell passage (random effect). –BCAA or –EAA did not affect GLUT-m (P = 0.114), GLUT-c (P = 0.248), and GLUT-t (P = 0.845). Each of –BCAA and –EAA equally increased the fraction of GLUT1 found in the cell membrane by 2-fold (P < 0.001). The results highlighted that extracellular BCAA deficiency did not likely affect the expression of GLUT1 but potentially enhanced the translocation of GLUT1 from cytoplasm to the cell membrane in BMEC.