Heparosan polysaccharide, known as a heparin precursor, can be used in drug delivery systems due to its good biocompatibility and anti-cancer effect. But few studies on the cellular uptake mechanism of heparosan polysaccharide-based nanocarrier have been investigated. Therefore, the intracellular trafficking and the uptake mechanism of heparosan-based micelles by different tumor cells were investigated in this study. Heparosan-cholesterol amphipathic conjugates (KC) were constructed and doxorubicin (DOX) was loaded to prepare DOX/KC micelles. Different cancer cells were selected to find out the influence on DOX/KC uptake. There was an obvious difference in cytotoxicity and cellular uptake of DOX/KC in various cancer cells. Interestingly, DOX/KC micelles exhibited the strongest cytotoxicity against MGC80-3 cells and displayed highly cellular uptake by B16 cells. The results of the uptake mechanism showed that the internalization of DOX/KC micelles into MGC80-3 cells and A549 cells was mainly through clathrin-mediated endocytosis and macropinocytosis, while micropinocytosis, clathrin-mediated endocytosis and clathrin/caveolae -independent multi-pathways all contributed to the uptake of micelles in B16 cells. Furthermore, after being internalized by MGC80-3 cells, DOX/KC could escape from the lysosome and simultaneously be transported into the nucleus and mitochondria resulting in the greatest cytotoxicity. These results indicate that heparosan-based drug delivery systems may have different uptake and subcellular distribution behavior in tumor cells, and they will achieve the maximum efficacy only in specific kind of cancers.