Background: HER2 over-expression/amplification, which accounts for roughly 15% of total biliary tract cancer (BTC) patients, has been identified as a druggable molecular target by recent genomic profilings, Trastuzumab is a humanized monoclonal antibody against HER2 that has been shown to be effective in patients with HER2-positive breast and gastric cancer, but it has not been studied prospectively in HER2-positive BTC. In the phase III ABC-06 trial, the FOLFOX regimen showed survival benefit as a second-line therapy of BTC. We report the result of a multi-institutional phase II trial of Trastuzumab plus modified-FOLFOX as a second- or third-line treatment for HER2-positive BTC (KCSG-HB19-14; NCT04722133). Methods: HER2-positive (defined as IHC3+ or IHC2+/ISH+ or ERBB2 gene copy number ≥6.0 by NGS) BTC (intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, gallbladder cancer and ampulla of vater cancer) patients who progressed on gemcitabine/cisplatin containing chemotherapy (1 or 2 previous chemotherapy lines permitted) were enrolled. Pts received trastuzumab 4mg/kg (after 6mg/kg load) D1, oxaliplatin 85mg/m² D1, Leucovorin 200mg/m² D1, 5-FU 400mg/m² bolus D1, and 5-FU 2400mg/m² infusion D1-2 every 2 weeks until unacceptable toxicities or disease progression. The primary endpoint was ORR per RECIST v1.1. Secondary endpoints included PFS, DCR, OS, safety, QOL and correlative biomarker exploration. Results: Total of 34 pts were treated with median follow up of 9.9 months, and 6 pts remained on treatment (treatment duration range: 1.0 to 14.7 months). The primary endpoint was met, with 29.4% (95%CI 15.1-47.5) ORR (PR n = 10), and 79.4% DCR. Median PFS was 5.1 months (95%CI 3.6-6.7) and median OS was not reached (95%CI 7.1-NR; 12-months OS rate 50.6%, 95%CI 29.3-63.6). Pts with HER2 IHC3+ (n = 23, 67.6%) showed tendency for better PFS compared to pts with HER2 IHC 2+/ISH+ (median 5.5 vs 4.9 months, HR 0.52, 95%CI 0.23-1.16). Pts with HER2 3+ tumor cell proportion ≥30% (n = 10) by an artificial intelligence-powered automated HER2 IHC analyzer (Lunit SCOPE HER2) showed significantly better PFS compared to pts without (median 6.67 vs 4.87 months, HR 0.33 95%CI 0.13-0.88). Targeted-panel sequencings were done with tumor tissues from 32 pts and tissue HER2-amplification by NGS did not confer better survival. Treatment-related AE (≥G3) occurred in 29 pts (85.3%) including 19 pts (55.9%) with neutropenia G3-4 and 4 pts (11.8%) with peripheral neuropathy G3-4. No pt showed cardiac AE nor treatment-related study discontinuation. Conclusions: For HER2-positive BTC, 2nd- or 3rd-line trastuzumab plus FOLFOX exhibited a promising efficacy with acceptable toxicity, warranting further investigations. Targeted NGS analyses with ctDNAs from pre-treatment and post-progression liquid biopsies are ongoing. Clinical trial information: NCT04722133.