Caveolin-3 is a member of the caveolin family of proteins that is primarily expressed in striated muscle cell types (skeletal and cardiac). Here, we show that an approximately 80-kDa protein specifically co-immunoprecipitates with caveolin-3 expressed in differentiated skeletal C2C12 myotubes. Microsequence analysis of this approximately 80-kDa polypeptide revealed its identity as a key regulatory enzyme in the glycolytic pathway, namely phosphofructokinase-M (PFK-M). Pulse-chase experiments demonstrate that PFK-M associates with caveolin-3 with a significant time lag after the biosynthesis of PFK-M. In addition, we show that this interaction is (i) highly regulated by the extracellular concentration of glucose and (ii) can be stabilized by a number of relevant intracellular metabolites, such as fructose 1,6-bisphosphate and fructose 2,6-bisphosphate, which are known allosteric activators of PFK. While the bulk of these experiments were performed in C2C12 cells, identical results were obtained using mouse skeletal muscle extracts. Taken together, our results suggest that glucose-dependent plasma membrane recruitment of activated PFK-M by caveolin-3 could have important implications for understanding the mechanisms that regulate energy metabolism in skeletal muscle fibers.