Objectives: Skeletal muscle cells are responsible for 80-90% of the insulin-induced glucose uptake in the body. Insulin signaling in skeletal muscle results in the targeted trafficking of glucose transporter type 4 (GLUT4) onto the cell membrane, enabling glucose uptake. Insulin resistant cells show defects in insulin-induced GLUT4 exocytosis. The eight-protein exocyst complex has an essential role in the insulin-induced exocytosis of GLUT4 vesicles in cultured adipocytes but it is not known if the exocyst-mediated molecular mechanism is conserved in other, insulin-responsive tissues, such as the skeletal muscle. We hypothesized that the exocyst complex is essential for the insulin-induced exocytosis of GLUT4-containing vesicles in skeletal muscle as well and that the exocyst is a master regulator of glucose homeostasis in insulin-responsive tissues. Methods/Results: We have generated a tamoxifen-inducible skeletal muscle-specific knockout mouse strain of exocyst central subunit Exoc5 (Exoc5-SMKO) to assess the exocyst’s role in glucose homeostasis in vivo. Exoc5 knockout does not affect grip strength, motor coordination or locomotor activity levels in these animals. Both male and female Exoc5-SMKO mice present with elevated fasting glucose levels, as compared to control littermates. Glucose tolerance testing revealed an impaired glucose clearance in Exoc5-SMKO mice, while insulin tolerance, fasting insulin levels, and A1C levels were similar between knockouts and controls. Conclusion: Our findings suggest that Exoc5 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. L.T. Carter: None. A.M. Wong: None. M.W. Pitts: None. R.K. Villiger: None. M. Young: None. B. Fogelgren: None. N. Polgar: None. Funding National Institute of General Medical Sciences (5P20GM113134)