Dysregulation of EGFR expression and signaling is well documented to contribute to disease progression and metastasis in many types of cancer including breast cancer. EGF-stimulated EGFR activation leads to receptor internalization and endocytic degradation to control EGFR-mediated signaling. This process is frequently deregulated in cancer cells, leading to increased EGFR expression and mitogenic signaling. Here, we demonstrate that Bif-1, a tumor suppressor, plays a role in EGFR endocytic degradation and chemotactic migration in MDA-MB-231 breast cancer cells. Our data reveal that suppression of Bif-1 expression delays EGFR degradation and sustains Erk1/2 activation in response to EGF stimulation. Mechanistically, loss of Bif-1 sequesters internalized EGF in Rab5-positive endosomes and delays EGFR trafficking to lysosomes. Recruitment of Rab7 to EGF-positive vesicles and the activation of Rab7 are impaired in Bif-1 knockdown cells. Additionally, intracellular pH and the localization of acidic vesicles are altered by suppression of Bif-1. Furthermore, inhibition of Bif-1 increases chemotactic cell migration in response to EGF or serum, which correlates with prolonged cytoskeletal reorganization. Importantly, the effect of Bif-1 on EGF-induced cell migration is abolished by gefitinib, an EGFR-specific inhibitor. Taken together, these data suggest a novel function for Bif-1 as a suppressor of breast cancer cell migration by promoting EGFR degradation through the regulation of endosome maturation.