Purpose: Postmenopausal women with estrogen receptor-positive breast cancers often respond initially to tamoxifen or aromatase inhibitor therapy. Resistance to these treatments usually develops within 12 to 18 months. Clinical studies have demonstrated that high-dose estrogen can induce regression of these endocrine-resistant tumors. However, side-effects of high-dose estradiol (E 2 ) or diethylstilbestrol (DES) limit their usage. Estetrol (E 4 ) is the most abundant estrogen during pregnancy and has a long half-life and a low potential for side-effects. Estetrol might then provide benefits similar to DES on tumor regression but with lesser toxicity. Methods: In this study, we systematically evaluated the effects of E 4 on cell proliferation and apoptosis in wild-type MCF-7 and long-term estrogen-deprived (LTED) MCF-7 cells and compared its effects with E 2 and estriol (E 3 ). Results: Estetrol induced apoptosis in LTED cells but stimulated growth of MCF-7 cells at concentrations from 10 −11 to 10 −8 M. These effects of E 4 are similar to those of E 2 but require much higher doses. Differing from E 2 , E 4 at 10 −12 M induced apoptosis in MCF-7 cells and another pregnancy estrogen, E 3 , acted similarly. No antagonistic effect of E 4 or E 3 against E 2 occurred when they were combined. Conclusions: The pro-apoptotic effects of E 4 and E 3 on LTED cells and at low doses on MCF-7 cells indicate that these steroids could be used as therapeutic agents for endocrine-resistant or sensitive breast cancer.