Background: Eribulin is a non-taxane microtubule inhibitor approved in China for the treatment of patients with advanced breast cancer who progress after ≥2 lines of chemotherapy. Eribulin was shown to improve overall survival (OS) in various subgroups of patients with metastatic breast cancer (MBC) who were pretreated with an anthracycline and taxane. Furthermore, combination chemotherapy with eribulin plus gemcitabine led to a similar progression free-survival (PFS) benefit, with less neurotoxicity, as paclitaxel plus gemcitabine in patients with MBC who had not received prior cytotoxic chemotherapy for metastatic disease. However, the effect of eribulin plus gemcitabine on PFS in second-line or beyond remain unclear. Methods: This phase II, open-label, single-arm study was conducted at 13 institutions in China (NCT05263882). Patients with histologically confirmed HER2-negative MBC who had received at ≥1 prior taxane-containing chemotherapeutic regimen for advanced disease and had received anthracycline-containing regimens in the adjuvant setting, were enrolled between Nov 2021 and Oct 2022. Eligible patients received eribulin 1.4 mg/m² and gemcitabine 1.0 g/m² intravenously on days 1 and 8 of a 21-day cycle. Efficacy outcomes included PFS, objective response rate (ORR), and disease control rate (DCR), evaluated using RECIST v1.1. Adverse events (AEs) were graded according to The National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 5.0. Results: In total, 54 patients were included; 39 (72.2 %) were HR+/HER2- and 15 (27.8 %) were triple negative, the median age was 49.5 years (range: 31-70) and sites of metastasis were the liver (57.4%), lung (37.0%), bone (59.3%), brain (11.1%) and lymph nodes (53.7%). The median number of previous chemotherapy lines was 3 (range: 2-7): eribulin plus gemcitabine was used in second-line for 15 (27.8%) patients, third-line for 20 (37.0%) patients, fourth-line for 7 (13.0%) patients and ≥fifth-line in 12 (22.2%) patients. The median PFS was 7.7 months. The ORR was 40.7% and the DCR was 66.7%. Best overall responses and PFS rates at 3 and 6 months are shown. The most common grade 3-4 AEs were hematological: Leukopenia (35.2%), anemia (18.6%) and thrombocytopenia (9.4%). No treatment-related death was reported. Conclusions: Eribulin plus gemcitabine is effective in heavily pretreated patients with HER2-negative MBC with a predictable, manageable safety profile.Clinical trial information: NCT05263882.