Background We evaluated the effectiveness of gemcitabine and paclitaxel therapy in patients with metastatic urothelial carcinoma for whom two lines of sequential chemotherapy had been unsuccessful. Methods A total number of 105 patients who had previously received first-line chemotherapy consisting of gemcitabine and cisplatin or carboplatin, were treated with second-line gemcitabine and docetaxel therapy between June 2006 and May 2015. Of these patients, 15 with an Eastern Cooperative Oncology Group Performance Status of 0 or 1 were administered gemcitabine and paclitaxel as third-line treatment from 2013 after failure of the second-line therapy. For each 21-day cycle, gemcitabine (1000 mg/m 2 ) was administered on days 1, 8, and 15, and paclitaxel (200 mg/m 2 ) on day 1. Patients were assessed for each cycle and any adverse events were noted. Furthermore, a Short Form Health Survey questionnaire was used to assess each patient's quality of life. Results Third-line gemcitabine and paclitaxel treatment cycles were undertaken for a median of four times (range 2-9). The disease control rate was 80.0%. After second-line gemcitabine and docetaxel therapy was completed, median progression-free survival and median overall survival were determined as 9.8 and 13.0 months, respectively. The only prognostic factor for overall survival, as determined by univariate and multivariate analyses, was third-line gemcitabine and paclitaxel therapy. Neutropenia (66.7%) and thrombocytopenia (53.3%) were noted as the grade 3 treatment-related toxicities. After two cycles of third-line gemcitabine and paclitaxel therapy, the pre- and post-treatment quality of life scores did not differ significantly. Conclusions Results demonstrate that third-line combination therapy using gemcitabine and paclitaxel is a feasible option for metastatic urothelial carcinoma patients.