Intracellular trafficking is a critical process for cell physiology. Previous extensive studies employing biochemical and molecular biological approaches have provided qualitative information about intracellular trafficking, but we have little quantitative information due to technical limitations of these assays. We therefore developed a novel method for quantifying intracellular trafficking based on single molecule imaging with Quantum dot (QD) fluorescent nanocrystals and quantitatively described the trafficking properties of some recycling proteins. We herein first describe how to label intracellular molecules with QD which has no cell permeability and how to quantify intracellular trafficking, and then we detail the development of a novel experimental system allowing multi-color simultaneous single molecule imaging for analyzing the relationships of intracellular trafficking activities among multiple molecules having distinct trafficking properties. Finally, we document how we confirmed the reliability of our system by simultaneously analyzing the intracellular movements of two recycling protein, GLUT4 glucose transporter and transferrin receptor. Since impairment of intracellular trafficking has critical etiological roles in various late-onset diseases such as type 2 diabetes, our novel imaging system may be a powerful tool for developing next-generation biomedical devices for diagnostics and medical treatment based on intracellular trafficking.