Background: The group of HER2-negative accounts for 70-80% of breast cancer and effective treatments for heavily pretreated patients with HER2-negative metastatic breast cancer are urgently needed. Irinotecan is a semi-synthetic water-soluble camptothecin derivative which can be used as a chemotherapeutic agent for HER2-negative breast cancer after failure of anthracycline and taxanes treatments. Anlotinib is a novel multitarget tyrosine kinase inhibitor targeting VEGFR, PDGFR, FGFR, and c-Kit. This study aimed to evaluate the efficacy and safety of irinotecan combined with anlotinib in second-line and above treatment of HER-2 negative advanced breast cancer. (ChiCTR2000037448) Methods: This open-label, single-arm, phase II study enrolled women aged 18 years and older with HER2-negative breast cancer who underwent ≥1 line chemotherapy, All patients were treated with irinotecan (60mg/m2, administered intravenously on Days 1 and 8 of each 21 day cycle) and anlotinib (12mg, qd, administered orally on Days 1-14 of each 21 day cycle). The primary endpoint was objective response rate (ORR). Secondary endpoints were progression-free survival (PFS), disease control rate (DCR), overall survival (OS) and safety. Results: From November 2020 to November 2022, 14 patients were enrolled in this study. Median follow-up was 10.8 months (95% CI 4.3-17.3). Median PFS was 5.9 months (95% CI 2.0-9.8). Of all the patients whose efficacy could be evaluated (13/14), no patient achieved complete response (CR); 3(23.07%) and 8(61.54%) patients got partial response (PR) and stable disease (SD), respectively. ORR was 23.1% (95% CI 5.0-53.8) and DCR was 84.6% (95% CI 54.6-98.1). OS has not reached. The major treatment-related adverse events (incidence≥10%) were fatigue (42.86%), hypertension (42.86%), neutropenia (28.57%), hand foot syndrome (21.43%) and myelosuppression (14.29%). There is no grade 3/4 adverse events occurred.28.57% (4/14) of patients had dose reductions. Conclusions: The combination of irinotecan and anlotinib showed better treatment response and tolerable toxicity in the treatment of second-line and above patients with HER2-negative metastatic breast cancer. Further studies enrolling more patients are still needed. Clinical trial information: ChiCTR2000037448.