Abstract: To investigate the EHD2 regulation of epithelial cell polarity and its mechanism, and to understand the molecular and cell biological significance of disordered EHD2 gene expression in breast cancer. Methods: The 2D and 3D culture of immortalized normal mammary epithelial cells were used as models, and the exogenous expression system of EHD2 mutant controlled by a tetracycline response element was established. Fluorescence microscopy and confocal laser scanning microscope imaging were used to observe the microfilament assembly before and after EHD2 mutant expression, and the effects on acini formation in Matrigel. Synthetic judgment of the molecular and cytologic effects of EHD2 protein in regulating epithelial cell polarity was carried out. Results: The induced EHD2 mutant expression significantly affected the rearrangement of the cell cytoskeleton in the immortalized breast epithelial cells. Dense structure of the microspikes generated on cell surface, orientation arrangement of the β-actin filament arrays inside the cell vanished, and the acini formation in 3D Matrigel, with invasive cells emerging into the matrix, was demolished. Conclusion: EHD2 mutation may result in cytoskeletal r-arrangement and polarity change in breast epithelial cells. EHD2 may possibly be one of the key mechanisms in breast tumorigenesis.