Amrubicin is a synthetic anthracycline which has been shown in preclinical studies to have broad-spectrum anti-tumor activity and a lower potential for cardiotoxicity as compared to doxorubicin. We conducted a phase 1/2 trial of single-agent amrubicin as second- or third-line treatment for women with metastatic breast cancer. Women with metastatic HER2-negative breast cancer who had normal cardiac function and measurable disease, received intravenous (IV) amrubicin every 3 weeks. Prophylactic treatment with granulocyte colony-stimulating factors (G-CSFs) was recommended. Escalating amrubicin doses were administered in a 3 + 3 design in the phase 1 portion to determine the maximum tolerated dose. Achievement of a median PFS ≥4.5 months would warrant further development of amrubicin in this setting. Seventy-eight women (median age 58 years) were treated (phase 1, 15 patients; phase 2, 63 patients). An amrubicin dose of 110 mg/m(2) every 3 weeks was selected as the phase 2 dose, and 66 patients were treated. Twelve of 66 patients (18%) achieved objective response, and the clinical benefit rate was 42%. Median PFS was 4 months (95% CI 2.5, 5.8). Neutropenia was the most common grade 3/4 toxicity, observed in 29 patients (44%). One patient experienced an asymptomatic transient left ventricular ejection fraction decline (grade 3). Although the study did not meet the predefined PFS, amrubicin was well tolerated at 110 mg/m(2) IV when administered every 3 weeks with prophylactic G-CSF, and was an active second- or third-line treatment for metastatic HER2-negative breast cancer.