Abstract: To study the endocytic protein EHD2 as a potential regulator of cell proliferation, adhesion, and migration in the immortalized non-cancerous breast epithelial cell line HBL100, and to reveal the mechanism of breast cancer development. Methods: Data mining was used to analyze the EHD2 gene expression from the published genome-wide microarray data of breast cancer tissue, and the relationship between the levels of EHD2 expression and tumor progression. shRNA interfered with EHD2 protein expression in the immortalized normal breast epithelial cell line HBL100. Western blot analysis was used to verify the knockdown efficacy. The effects of EHD2 on cell functions and abilities were determined using assays for cell proliferation, adhesion, and chemotaxis. Results: The gene expression chip datasets from separate cohort studies showed and that EHD2 expression was inversely correlated with breast cancer progression. A clone of HBL100 cell line with EHD2 RNA interference exhibited reduced substrate adhesion, and increased epidermal growth factor–induced chemotaxis and proliferation. After shRNA inference of the EHD2 expression in the HBL100 cell line, both the rate of cell growth by 2D culture and that of cell migration by EGF induction were increased. There were significant differences between the two ( P < 0.005 ). The cell adhesion ability was decreased ( P < 0.05 ). Conclusion: EHD2 participates in regulating the proliferation, adhesion, and migration of HBL100 cells. The low expression level of EHD2 gene in breast carcinoma suggests that EHD2 might be inhibitory to breast tumor progression, and may possibly be a new indicator and new target in the molecular diagnosis and targeted therapy of breast cancer.