Purpose To date, it is not clear which anticancer agent is useful in combination with trastuzumab and pertuzumab As the first and second selective regimens for advanced or metastatic breast cancer (AMBC), this multicenter, open-label, phase II trial (JBCRG-M03: UMIN000012232) presents a prespecified analysis of eribulin in combination with pertuzumab and trastuzumab. Methods We enrolled 50 patients with no or single prior chemotherapy for HER2-positive AMBC during November 2013-April 2016. All patients received adjuvant or first-line chemotherapy with trastuzumab and a taxane. The treatment comprised eribulin on days 1 and 8 of a 21-day cycle and trastuzumabplus pertuzumab once every 3 weeks, all administered intravenously. While the primary endpoint was the progression-free survival (PFS), secondary endpoints were the response rate and safety. Results Of 50 patients, 49 were eligible for safety analysis, and the full analysis set (FAS) included 46 patients. We treated 8 (16%) and 41 (84%) patients in first- and second-line settings, respectively. While 11 patients (23.9%) had advanced disease, 35 (76.1%) had metastatic disease. The median PFS was 9.2 months for all patients [95% confidence interval (CI): 7.0-11.4]. In the FAS, 44 patients had the measurable lesions and the complete response rate (CR) was 17.4%, and partial response rate (PR) was 43.5%. The grade 3/4 adverse events were neutropenia (5 patients, 10.2%), including febrile neutropenia (2 patients, 4.1%), hypertension (3 patients, 6.1%), and other (1 patient). The average of the left ventricular ejection fraction did not decline markedly. No symptomatic left ventricular systolic dysfunction was observed. Conclusions In patients with HER2-positive AMBC, eribulin, pertuzumab, and trastuzumab combination therapy exhibited substantial antitumor activity with an acceptable safety profile. Hence, we have started a randomized phase III study comparing eribulin and a taxane in combination with pertuzumab and trastuzumab for the treatment of HER2-positive AMBC. Trial registration ID: UMIN-CTR: UMIN000012232.