Metastasis is the major cause of morbidity and mortality in cancer. Recent studies reveal a role of chemotaxis in cancer cell metastasis. Epidermal growth factor receptors (EGFR) have potent chemotactic effects on human breast cancer cells. Lipid rafts, organized microdomain on plasma membranes, regulate the activation of many membrane receptors. In the current study, we investigated the role of lipid rafts in EGFR-mediated cancer cell chemotaxis. Our confocal microscopy results suggested that EGFR co-localized with GM1-positive rafts. Disrupting rafts with methyl-beta-cyclodextrin (mbetaCD) inhibited EGF-induced chemotaxis of human breast cancer cells. Supplementation with cholesterol reversed the inhibitory effects. Pretreatment with mbetaCD also impaired directional migration of cells in an in vitro "wound healing" assay, EGF-induced cell adhesion, actin polymerization, Akt phosphorylation and protein kinase Czeta (PKCzeta) translocation. Taken together, our study indicated that integrity of lipid rafts was critical in EGF-induced chemotaxis of human breast cancer cells.