Background: Currently there is no established standard treatment after failure of gemcitabine plus cisplatin (GemCis) for advanced biliary tract cancer (BTC). Based on the efficacy of FOLFIRINOX in advanced pancreatic cancer, which has histological and prognostic similarities with BTC, a Phase 2 study was conducted to determine whether FOLFIRINOX is effective and safe in BTC. Methods: Patients with BTC and an ECOG PS of 0/1 who had disease progression or unacceptable adverse events (AEs) after at least 3 cycles of GemCis were included. Patients received oxaliplatin 85 ml/m², irinotecan 180 mg/m², leucovorin 400 mg/m², fluorouracil bolus at 400 mg/m2, followed by fluorouracil continuous infusion of 2400 mg/m² over 46-hour every 2 weeks. This phase 2 study was conducted according to the two-stage Simon’s Design. Stage 2 was activated if at least 1 objective response rate (ORR) or 2 stable diseases were observed among 10 patients in stage 1 and a maximum of 3 patients had severe AEs within the first 6 weeks of treatment. If more than 4 patients required a dose reduction in stage 1, stage 2 was initiated with a standard dose reduction (fluorouracil bolus was omitted and irinotecan reduced to 140 mg/m²). Primary outcome was ORR per RECIST 1.1 and secondary outcomes were overall (OS), progression free survival (PFS), and safety profile. Results: Forty patients were screened and 30 patients were included between May 2016 and July 2018. Median age was 60 years and 63% of patients were males. In stage 1, 5 patients required a dose reduction within the first 6 weeks due to AEs, leading to initiation of stage 2 with modified FOLFIRINOX after inclusion of 10 patients. The partial response rate was 10% (3/30), disease control rate 67% and median OS and, PFS were 10.7 and 6.2 months, respectively. Most common grade 3/4 adverse events include neutropenia (50%), anemia (17%), diarrhea (13%), thrombocytopenia (10%), and deviated liver function tests (10%). Conclusions: This is the first Phase 2 study with modified FOLFIRINOX in BTC showing promising disease control rate, OS, and PFS, with an acceptable safety profile. Modified FOLFIRINOX is currently tested as a first-line treatment for patients with BTC in the ongoing randomized prospective Phase 2/3 AMEBICA trial (NCT02591030). Clinical trial information: NCT02456714