Insulin increases glucose uptake in skeletal muscle and adipocytes and thus facilitates the disposal of ~90% of glucose after a meal. The increased glucose uptake is achieved through the translocation of the glucose transporter GLUT4 from an intracellular compartment to the cell surface. Recent studies have implicated two Rab GTPase-activating proteins (Rab GAPs), AS160 and Tbc1d1, in the regulation of glucose uptake and GLUT4 subcellular distribution. To determine the roles of the two Rab GAPs in GLUT4 subcellular distribution, glucose uptake, and whole body glucose and energy homeostasis, we characterized single AS160 and Tbc1d1 and double AS160/Tbc1d1 knockout (KO) mice. Our results with isolated skeletal muscles and adipocytes demonstrate that AS160 but not Tbc1d1 regulates glucose uptake in adipocytes, while AS160 and Tbc1d1 each regulate glucose uptake in specific skeletal muscles. Our results further show that AS160 or Tbc1d1 deletions lead to similar changes in glucose uptake, total GLUT4 and GLUT4 cell surface expression in the specific skeletal muscles, but AS160 deletion leads to different changes in glucose uptake and GLUT4 cell surface expression between skeletal muscle and adipocytes. At the whole body level glucose homeostasis is prominently regulated by AS160, while energy expenditure is primarily controlled by Tbc1d1. Our observations further show that the two Rab GAPs do not compensate for each other in the single knockout mice, and the double AS160/Tbc1d1 knockout mice combine the defects of the single knockout mice at both the cellular and the whole body level. In conclusion, our results suggest that AS160 and Tbc1d1, consistent with their differential tissue distribution, have distinct roles with regard to where they regulate glucose uptake. However, they may have similar functions with regard to how they regulate GLUT4 expression and translocation. Lastly, the two Rab GAPs differentially regulate whole body glucose and energy homeostasis.