Background: After first-line treatment with gemcitabine-based regimen, there is not standard second-line chemotherapy for patients with metastatic gallbladder cancer (GB) and cholangiocarcinoma (CC). The aim of this study was to evaluate the activity, progression free survival (PFS), and overall survival (OS) of the FOLFOX4 regimen as second-line chemotherapy. Methods: We performed a retrospective analysis of all metastatic GB and CC that were treated with gemcitabine (Gem) based first-line chemotherapy, and second-line FOLFOX4 chemotherapy (oxaliplatin 85 mg/m2, day 1; leucovorin 200 mg/m intravenously, days 1 and 2; 5-fluorouracil 400 mg/m intravenous bolus, days 1 and 2; and 600 mg/m in 22 h intravenously, continuous infusion days 1 and 2; every 14 days) in our Institution since 2003. The response rate was evaluated every 2 to 4 months according to the RECIST criteria, and PFS/OS were calculated according to the Kaplan-Meier method. Results: Eighty patients (n=40 GB; n=40 CC) received Gem based first-line treatment (42 Gem alone; 38 Gem + platinum). There was no survival difference between Gem or Gem+platinum treatment (161 versus 254 days; p=0.09). Response rate (complete and partial response) was 16% and stable disease was 31%. Ca19-9 at presentation was predictive for OS: <100U/ml =403 days versus >100 U/ml =223 days (p=0.025). Eighteen patients (22%; n=10 GB and n=8 CC) were treated with second-line FOLFOX4. In the second-line group, stable disease was achieved in 4 patients (22%), and 11 patients progressed (66%). The median DFS and OS were 96 and 138 days respectively. The median OS for patients that received one versus both lines of chemotherapy were 161 versus 400 days (p=0.009). Conclusions: FOLFOX4 has activity in second line chemotherapy for patients with metastatic GB and CC. A prospective study is warranted to confirm this observation.