Insulin triggers the translocation of glucose transporter GLUT4 to the plasma membrane. To understand the nature of the missing links between upstream insulin activated kinases and proteins of the GLUT4 translocation apparatus, the role of 80K-H was examined to test if it was one such missing link in live cells. Fluorescence correlation spectroscopy showed that the mobility of 80K-H was significantly decreased by insulin stimulation. This was dependent on the presence of PKCzeta and an intact binding site for PKCzeta. Insulin also increased the mobility of munc18c in an 80K-H- and PKCzeta dependent manner. These results indicate that insulin induces dynamic associations between PKCzeta, 80K-H, and munc18c and that 80K-H may act as a key signaling link between PKCzeta and munc18c in live cells.