To investigate the importance of the glucose transporter GLUT‐4 for muscle glucose uptake during exercise, transgenic mice with skeletal muscle GLUT‐4 expression approximately 30–60% of normal (CON) and approximately 5–10% of normal (KO) were generated using the Cre/Lox system and compared with wild‐type (WT) mice during approximately 40 min of treadmill running (KO: 37.7 ± 1.3 min; WT: 40 min; CON: 40 min, P = 0.18). In WT and CON animals, exercise resulted in an overall increase in muscle glucose uptake. More specifically, glucose uptake was increased in red gastrocnemius of WT mice and in the soleus and red gastrocnemius of CON mice. In contrast, the exercise‐induced increase in muscle glucose uptake in all muscles was completely abolished in KO mice. Muscle glucose uptake increased during exercise in both red and white quadriceps of WT mice, while the small increases in CON mice were not statistically significant. In KO mice, there was no change at all in quadriceps muscle glucose uptake. No differences in muscle glycogen use during exercise were observed between any of the groups. However, there was a significant increase in plasma glucose levels after exercise in KO mice. The results of this study demonstrated that a reduction in skeletal muscle GLUT‐4 expression to approximately 10% of normal levels completely abolished the exercise‐induced increase in muscle glucose uptake.