GLUT4 is sequestered in intracellular storage compartments in a basal state and is rapidly translocated to the cell surface in response to insulin stimulation. Regulation of GLUT4 distribution is key for maintaining whole-body glucose homeostasis. To investigate the complicated intracellular movement of GLUT4 vesicles and their interactions with organelles in detail, new probes suitable for long-term tracking of cellular events are required. In this study, we introduce for the first time quantum dots (QDs) as a superior probe into the research of the mechanisms of GLUT4 translocation. QDs are light-emitting semiconductor nanoparticles with unique optical and spectroscopic properties, such as broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. Owing to their remarkable photophysical properties and relatively small size, QDs are emerging as an alternative to conventional dyes for fluorescence-based applications. We have developed a procedure for labeling and imaging GLUT4 in live cells with streptavidin-conjugated quantum dot (QD-SA) and demonstrated that QDs contained in cytoplasm have no obvious negative influence on L6 cells. This study provides a sensitive, nontoxic, long-term imaging platform for observing the dynamics and regulated characteristics of GLUT4 transport.