Background: The current standard of care for patients with advanced biliary tract cancer (BTC) is a palliative chemotherapy. Although the survival benefit of first-line chemotherapy (CT1) including gemcitabine and cisplatin (GC) had been established in prospective randomized trials, the safety and efficacy of second-line chemotherapy (CT2) have yet to be elucidated. Methods: Consecutive advanced BTC patients receiving CT1 followed by CT2 between 2000 and 2016 were enrolled. We investigated factors associated with receiving CT2, treatment outcomes of CT2, and the prognostic factors for overall survival (OS) with CT2 to determine which patients could be expected to benefit from CT2. Results: Among 309 advanced BTC patients receiving CT1, including those with 108 intrahepatic bile duct cancer, 96 gallbladder cancer, 88 extrahepatic bile duct cancer and 17 papilla cancer, CT2 was given in 139 patients (45%). Compared to patients without CT2, younger patients (median; 67 vs. 70 years, p < 0.01) and patients with better PS (PS 0-1; 98% vs. 94%, p = 0.06), and those who achieved higher tumor response by CT1 (response rate; 19% vs. 7%, p < 0.01) had higher chance to receive CT2. Objective response rate and disease control rate of CT2 were 4% and 50%, respectively. Median progression-free survival (PFS) and overall survival (OS) from the beginning of CT2 were 2.8 and 7.7 months, respectively. Prognostic factors impacting OS with CT2 were PS (0 vs. 1-2, hazard ratio [HR] 1.28, p=0.05), tumor response to CT1 (PR-SD vs. PD, HR 1.62, p = 0.02), and CEA (<5.0 vs. ≥5.0 IU/mL, HR 1.52, p = 0.03). Conclusions: Although the efficacy of CT2 was modest at present, it may deserve to be considered in patients who maintained systemic condition in clinical practice. Further investigations are necessary both to to develop more effective regimens and to select patient who will benefit from CT2 in order to maximize the efficacy and to avoid unnecessary toxicity and expense.