Abstract: In this study, the endocytosis pathway of heparosan and its intracellular distribution were investigated in MCF-7 tumor cells and COS7 normal cells. The endocytosis inhibition and cellular probe location experiments showed that MCF-7 tumor cells took heparosan more efficiently and selectively than COS7 cells. The cellular uptake of heparosan was energy-dependent in both MCF-7 tumor cells and COS7 normal cells. Moreover, the major endocytosis pathway of heparosan into MCF-7 tumor cells was caveolin-mediated endocytosis and macropinocytosis. The internalized heparosan was mainly located in lysosomes of the cells.