Strategies to implement estrogen therapy for advanced estrogen receptor-positive (ER+) breast cancer are underdeveloped. Preclinical data suggest that cycling treatment with 17β-estradiol followed by estrogen deprivation can control tumor growth long-term. Postmenopausal women with advanced ER+/HER2- breast cancer with recurrence or progression on ≥ 1 antiestrogen or aromatase inhibitor (AI)-based therapy were eligible. Patients received 17β-estradiol (2 mg orally, three times a day) for 8 weeks followed by AI (physician's choice) for 16 weeks, alternating treatments on an 8-week/16-week schedule until disease progression. Patients then optionally received continuous single-agent treatment until a second instance of disease progression. Endpoints included 24-week clinical benefit and objective response per RECIST, and tumor genetic alterations. Of 19 evaluable patients, clinical benefit rate was 42.1% [95% confidence interval (CI), 23.1%-63.9%] and objective response rate (ORR) was 15.8% (95% CI, 5.7%-37.9%). One patient experienced a grade 3 adverse event related to 17β-estradiol. Among patients who received continuous single-agent treatment until a second instance of disease progression, clinical benefit was observed in 5 of 12 (41.7%) cases. Tumor ER (ESR1) mutations were found by whole-exome profiling in 4 of 7 (57.1%) versus 2 of 9 (22.2%) patients who did versus did not experience clinical benefit from alternating 17β-estradiol/AI therapy. The only two patients to experience objective responses to initial 17β-estradiol had tumor ESR1 mutations. Alternating 17β-estradiol/AI therapy may be a promising treatment for endocrine-refractory ER+ breast cancer, including following progression on CDK4/6 inhibitors or everolimus. Further study is warranted to determine whether the antitumor activity of 17β-estradiol differs according to ESR1 mutation status.