Acquired tamoxifen (TAM) resistance limits the therapeutic benefit of TAM in patients with hormone‐dependent breast cancer. The switch from estrogen‐dependent to growth factor‐dependent growth is a critical step in this process. However, the molecular mechanisms underlying this switch remain poorly understood. In this study, we established a TAM resistant cell sub line (MCF‐7/TAM) from estrogen receptor‐α (ER‐α66) positive breast cancer MCF‐7 cells by culturing ER‐α66‐positive MCF‐7 cells in medium plus 1 μM TAM over 6 months. MCF‐7/TAM cells were then found to exhibit accelerated proliferation rate together with enhanced in vitro migratory and invasive ability. And the estrogen receptor‐α36 (ER‐α36), a novel 36‐kDa variant of ER‐α66, was dramatically overexpressed in this in vitro model, compared to the parental MCF‐7 cells. Meanwhile, the expression of epidermal growth factor receptor (EGFR) in MCF‐7/TAM cells was significantly up‐regulated both in mRNA level and protein level, and the expression of ER‐α66 was greatly down‐regulated oppositely. In the subsequent studies, we overexpressed ER‐α36 in MCF‐7 cells by stable transfection and found that ER‐α36 transfected MCF‐7 cells (MCF‐7/ER‐α36) similarly exhibited decreased sensitivity to TAM, accelerated proliferative rate and enhanced in vitro migratory and invasive ability, compared to empty vector transfected MCF‐7 cells (MCF‐7/V). Real‐time qPCR and Western blotting analysis revealed that MCF‐7/ER‐α36 cells possessed increased EGFR expression but decreased ER‐α66 expression both in mRNA level and protein level, compared to MCF‐7/V cells. This change in MCF‐7/ER‐α36 cells could be reversed by neutralizing anti‐ER‐α36 antibody treatment. Furthermore, knock‐down of ER‐α36 expression in MCF‐7/TAM cells resulted in reduced proliferation rate together with decreased in vitro migratory and invasive ability. Decreased EGFR mRNA and protein expression as well as increased ER‐α66 mRNA expression were also observed in MCF‐7/TAM cells with down‐regulated ER‐α36 expression. In addition, blocking EGFR/ERK signaling in MCF‐7/ER‐α36 cells could restore the expression of ER‐α66 partly, suggesting a regulatory function of EGFR/ERK signaling in down‐regulation of ER‐α66 expression. In conclusion, our results indicated for the first time a regulatory role of ER‐α36 in up‐regulation of EGFR expression and down‐regulation of ER‐α66 expression, which could be an underlying mechanism for the growth status switch in breast tumors that contribute to the generation of acquired TAM resistance. And ER‐α36 could be considered a potential new therapeutic target in breast tumors which have acquired resistance to TAM.