Eosinophil cationic protein (ECP), a human RNAseA superfamily member, highly implicated in asthma pathology, is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In this study, we show that cell surface-bound heparan sulfate proteoglycans serve as the major receptor for ECP internalization. Removal of cell surface heparan sulfate by heparinases or reducing glycan sulfation by chlorate markedly decreased ECP binding to human bronchial epithelial Beas-2B cells. In addition, ECP uptake and associated cytotoxicity were reduced in glycosaminoglycan-defective cells compared with their wild-type counterparts. Furthermore, pharmacological treatment combined with siRNA knockdown identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and ADP-ribosylating factor 6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and phosphatidylinositol-3-kinase activities, and is compatible with the characteristics of raft-dependent macropinocytosis. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.