The rates of glucose transport and of glycolysis and the expression of the glucose transporters GLUT-1 through GLUT-4 were measured in T47D human breast cancer cells that underwent differentiation by retinoic acid. Glucose transport was found to be the rate-limiting step of glycolysis in control and differentiated cells. The transporters GLUT-1, GLUT-3, and GLUT-4 were present in the cell membrane and in the cytoplasm, and GLUT-2 was present solely in the cytoplasm. Differentiation led to a reduction in GLUT-1 and to an increase in cytoplasmic GLUT-2 and GLUT-3 with no change in GLUT-4. Differentiation also caused a reduction in the maximal velocity of glucose transport by ~40% without affecting the Michaelis-Menten constant of glucose transport. These changes did not alter the steady-state concentration of the phosphate metabolites regulating cell energetics but increased the content of phospholipid breakdown phosphodiesters. In conclusion, differentiation of human breast cancer cells appears to be associated with decreased glycolysis by a mechanism that involves a reduction in GLUT-1 and a slowdown of glucose transport.