We aimed to assess the efficacy of second-line fluoropyrimidine-based chemotherapy in patients with advanced biliary tract cancer (BTC) after failure of gemcitabine plus cisplatin (GEMCIS). We retrospectively examined patients with histologically documented advanced BTC who received first-line GEMCIS between December 2010 and June 2015. Among 748 patients treated with first-line GEMCIS, 321 (43%) subsequently received fluoropyrimidine-based second-line systemic chemotherapy. Fluoropyrimidine monotherapy and fluoropyrimidine-platinum combination were used in 255 and 66 patients, respectively. In patients with measurable disease, the overall response rate (ORR) was 3% and disease control rate was 47%. After a median follow-up of 27.6 months (range, 0.9-70.4 months), the median progression-free survival (PFS) and overall survival (OS) were 1.9 months (95% confidence interval (CI), 1.6-2.2) and 6.5 months (95% CI, 5.9-7.0), respectively. The ORR was significantly higher in patients who received fluoropyrimidine-platinum combination compared with those who received fluoropyrimidine alone (8 vs 1%, P=0.009), although the PFS (P=0.43) and OS (P=0.88) did not significantly differ between these groups. Fluoropyrimidine-based chemotherapy was modestly effective as a second-line chemotherapy for advanced BTC patients after failure of GEMCIS. Fluoropyrimidine-platinum combination therapy was not associated with improved survival outcomes, as compared with fluoropyrimidine monotherapy.