Objectives: Skeletal muscle cells are responsible for 80-90% of the insulin-induced glucose uptake in the body. Insulin activation of muscle cells triggers a signaling cascade that results in the exocytosis of membrane-bound glucose transporter type 4 (GLUT4) to the plasma membrane. The eight-protein complex called the exocyst is recognized as having an essential role in the insulin-induced exocytosis of GLUT4 vesicles in cultured adipocytes. We hypothesize that Sec10, a central component of the exocyst complex, is essential for the insulin-induced exocytosis of GLUT4 vesicles in skeletal myoblasts and that the exocyst is a master regulator of glucose homeostasis in metabolic tissues. Methods/Results: To analyze exocyst-mediated intracellular trafficking in skeletal muscle in vitro, we used L6 GLUT4-myc rat skeletal myoblasts, and CRISPR/Cas9 to create Sec10 knockout (Sec10-KO) clones from these cells. Immunofluorescent staining shows co-localization of exocyst Sec10 and GLUT4 upon insulin signal in L6 myoblasts. Cellular fractionation reveals that GLUT4 delivery to the plasma membrane in response to insulin is impaired in Sec10-KO cells. Also, glucose uptake rates are significantly decreased in Sec-10-KO L6 myoblasts compared to wild type cells upon insulin stimulus. We have also generated a tamoxifen-activated skeletal muscle-specific Sec10-knockout mouse strain to assess the exocyst’s role in glucose homeostasis in vivo. Sec10 knockout mice demonstrate impaired glucose tolerance compared to littermate controls. Conclusion: Based on our findings, Sec10 and the exocyst are necessary for insulin stimulated glucose uptake in skeletal muscle. Ongoing work will further investigate the molecular mechanism of exocyst-mediated GLUT4 trafficking in skeletal muscle. Disclosure B. Fujimoto: None. A. Lee: None. A.M. Wong: None. L.T. Carter: None. B. Fogelgren: None. N. Polgar: None.