Abstract: Objective To investigate the efficacy, adverse effect and survival of continued use of trastuzumab across multiple lines plus different chemotherapy regimens on HER2 positive advanced breast cancer patients. Methods Seventy-one patients with HER2 positive advanced breast cancer were treated with trastuzumab across multiple lines plus different chemotherapy regimens from Jan., 2009 to June, 2014. All 71 patients received trastuzumab as a first-line therapy. After disease progression, trastuzumab was administered as a second-line therapy on 30 patients, 19 patients were treated with trastuzumab as a third-line therapy beyond disease progression again. We evaluated the clinical efficacy, side effect and outcome. Results In the first-, second-, third-line treatment, there was no statistical difference in response rate(RR) or clinical benefit rate (CBR) between trastuzumab combined with taxane drugs and non-taxane drugs (P＞0.05). The median progression free survival(PFS) and overall survival(OS) were 14, 9, 4 months and 26, 39, 53 months in the first-, second-, third-line treatment. The total PFS was 11 months and the total OS was 36 months. The PFS in the first-line therapy was longer than those in the second- and third-line therapies(P=0.000). The OS of continued use of trastuzumab to the third-line therapy was longer than that only to the firstline therapy(P=0.008). The 1-, 2- and 3-year survival rates were 88%, 66% and 39%. Fourteen of the 71 patients had 17 cardiac events; one patient was terminated trastuzumab therapy because of a left ventricular ejection fraction(LVEF) decreasing to 48%, but no fatal cardiac event was reported. The number of lymphatic metastasis, brain metastases, different treatment lines and the PFS of the first-line treatment were concerned with OS in Log rank single factor analysis(P=0.026, P=0.042, P=0.028, P=0.005). The different treatment lines, brain metastases, DFS and the PFS of the first-line treatment were independently prognostic factors for OS in Cox proportional hazards model analysis (P=0.004, P=0.021, P=0.018, P=0.000). Conclusion For patients with HER2-positive advanced breast cancer, trastuzumab across multiple lines combined with different chemotherapy is superior to those without continued trastuzumab treatment. Patients are benefit continually from trastuzumab treatment beyond disease progression. The administration of trastuzumab across multiple lines combined with chemotherapy is effective, well tolerated and worthy of further study.