Over 130 million Americans suffer from the devastating consequences of type 2 diabetes or prediabetes. Skeletal muscle accounts for up to 85% of insulin-induced blood glucose clearance, and insulin resistance for muscle glucose uptake is an essential defect for type 2 diabetes. One exercise bout can enhance subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle, but the mechanisms have remained elusive. The long-range goal of our research is to fully understand the molecular, cellular, and physiological events responsible for this significant health benefit. Our recent research using a unique Akt substrate of 160 kDa-knockout (AS160-KO) rat model revealed that expression of AS160 (a key regulator of GLUT4 glucose transporter localization) is essential for the elevated postexercise ISGU. We aim to identify specific Rab proteins that collaborate with AS160 to regulate postexercise ISGU by skeletal muscle. We will also perform other experiments that pursue advancement of knowledge related to AS160’s regulation of subcellular GLUT4 localization in muscle postexercise. Finally, our earlier research revealed that although AS160 expression is essential for elevated postexercise ISGU in both sexes, AS160 phosphorylation of several key sites is required only in male rats. We will perform experiments that aim to identify mechanisms that underlie this sex-related difference in the mechanisms responsible for greater postexercise ISGU, a major health benefit.