Background: Advanced biliary cancers respond poorly to chemotherapy and have a dismal survival. Currently gemcitabine-cisplatin (GC) is the standard of care for patients with advanced disease. The efficacy of second line treatment options is not well-defined. We retrospectively evaluated outcomes of patients undergoing second line treatment for advanced biliary cancers at Mayo Clinic. Methods: After obtaining approval from the institutional board review (IRB), we identified 42 patients with advanced and metastatic biliary cancers that progressed after first line (GC) and received second line treatment from 2007-2014. All patients had adequate follow up data. JMP software was used for statistical analysis. Kaplan-Meier method and log-rank tests were used for survival analysis, and multivariate cox proportional hazards model was used to evaluate the prognostic effect of pertinent clinical variables. All tests were two sided and a P value of < 0.05 was considered significant. Results: The median age at diagnosis was 61.5 (range 26-87). The most common regimens used for second line settings were 5-FU-based (69%) followed by gemcitabine-based regimens (19%). The overall response rate (ORR) was 9.1%, while the disease control rate (DCR; stable disease and partial response) was 33%. The median overall survival (mOS) for the entire cohort from first line was 16.8 months (95% CI 12.7-21.7) and the progression-free survival (PFS) was 9.16 months (95% CI 7.3-14.7). From second line start the mOS was 10.4 months (95%; CI 7.3-23.7), and PFS was 2.8 months (95% CI 2.3-4.0). 73% of patients who received second line therapy were alive at 6 months and 50% at 12 months. 25 patients received third line treatment after progression. An univariate and multivariate analysis were performed including age, sex, stage at diagnosis, regimen used, CA19-9 and total body weight loss > 5%, but no significant prognostic factors were identified. Conclusions: Patients with biliary cancers who progress after first line treatment have poor prognosis, but may benefit from second line regimens. 5-FU based regimens were the most commonly used in the second line setting, and 73% of patients who received second line therapy were alive at 6 months.