Estrogen Receptor alpha (ER alpha) affects the morphology of tumors, which is closely related to the biomechanical properties and the cytoskeletal proteins. In recent years, researchers have found that biomechanical properties and cytoskeletal proteins are closely related to the occurrence and development of tumors and that biomechanical properties can be used as markers for tumor development and drug resistance. The relationship between ER alpha expression status and biomechanical properties, cytoskeletal proteins is not known. In this study, we found that tamoxifen-resistant breast cancer cells (MCF-7/TamR) altered cell morphology and lacked of ER alpha expression during the process of the Tamoxifen resistance induction. To determine whether this change was influenced by ER alpha expression, we transiently constructed another ER alpha depleted model with ER alpha siRNA (MCF-7/ER alpha siRNA) and used atomic force microscope (AFM) to detect morphological and biophysical changes. The results indicated that the roughness and Young's modulus of ER alpha expression depleted cells were significantly increased, accompanied by rearrangement of the cytoskeletal proteins (F-actin, FLNA, alpha-tubulin) and the cytoskeletal regulatory protein Rho (Rac1, CDC42) decreased. Our results have demonstrated that ER alpha depletion affects the biomechanical properties of breast cancer cells, which are related to cytoskeletal protein rearrangement and Rho protein decreased. (c) 2020 Elsevier Inc. All rights reserved.