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The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions. In the present study, we examined whether the uPAR plays any role in the regulation of glucose metabolism. The experiments were performed using male wild-type (uPAR+/+) and uPAR knockout (uPAR−/−) C57BL/6J mice. The blood glucose levels after the intraperitoneal injection of glucose were significantly decreased in uPAR−/−mice compared with uPAR+/+mice. On the other hand, there were no differences in the insulin secretion induced by glucose injection and the reactivity of insulin between uPAR+/+and uPAR−/−mice. The expression levels of glucose transporter 2 (GLUT2) in the liver and GLUT4 in the skeletal muscles from the uPAR−/−mice were significantly increased compared with those of the uPAR+/+mice. In addition, we found that the level of phosphorylation of AMP-activated protein kinase in skeletal muscles and myoblasts from the uPAR−/−mice increased compared with those in uPAR+/+mice. These data suggest that the increase in the GLUT2 and GLUT4 expression and the activation of AMP-activated protein kinase by uPAR deficiency enhances the glucose intake. These findings therefore provide new insights into the role of uPAR in the glucose metabolism.