Dietary glucose is mainly taken up by skeletal muscle, primarily through glucose transporter GLUT4. We recently identified a cohort of proteins that co-purify with GLUT4 upon immunoprecipitation and mass spectrometry analysis (J Proteome Res, 2006). Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated from total cell lysates via the myc epitope. One attractive co-precipitating protein is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme involved not only in glycolytic ATP generation but also recently shown to interact with membranes and the cytoskeleton. Here, we applied in vitro pull-down assays using chimeric Glutathione S-Transferase (GST) fusion proteins linearly encompassing the cytosol-directed regions of GLUT4 or GLUT1 (N and C terminus and cytoplasmic loop), or the cytoplasmic loops alone, each bound to Sepharose beads. Commercial, purified human GAPDH, or endogenous GAPDH from L6 cell lysates bound to GST-GLUT4-cytosolic gt; GST-GLUT4-loop gt; GST-GLUTl-loop. GST alone did not pull down GAPDH. Addition of GLUT4 C-terminus peptide, but not cytoplasmic loop or N-terminal peptides, decreased GAPDH pull-down by cyto-GLUT4. These results suggest that GAPDH binds directly to GLUT4 cytosolic regions, and that the GLUT4 C-terminus contributes to such binding. Immuno-dot blot assay also showed that GAPDH binds to the purified GST-GLUTs constructs but not to GST alone. Seeking the physiological effect of the GAPDH-GLUT interaction, we knocked-down GAPDH via siRNA. A 60-70% reduction in GAPDH levels had no effect on glucose uptake or surface GLUT4 levels with or without insulin stimulation. Interestingly, however, the levels of Hexokinase-II coprecipitating with GLUT4myc from L6 cell lysates, increased upon GAPDH knock-down. These results indicate that GAPDH interacts directly with GLUT4 and that, this interaction might be part of a glycolytic metabolon, such that GAPDH may compete with Hexokinase-II. This project has the potential to reveal new mechanisms of regulation of glucose flux and utilization in muscle.