Background: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate composed of trastuzumab and DM1 (an anti-microtubule agent derived from maytansine) that is approved for the treatment of HER2-positive metastatic breast cancer. HER2-overexpression rate in biliary tract cancer (BTC) is reported as approximately 10%, and HER2-targeted therapy may enhance therapeutic efficacy in HER2-positive BTC. In this study, we investigated the antitumor activity of T-DM1 in several BTC cell lines and xenograft models. Methods: HER2 expression was detected by flow cytometry and Western blotting in 17 BTC cell lines. The cell growth inhibition activity of T-DM1 and DM1 was examined by quantifying the DNA with Hoechst 33258 nucleic acid stain. In vivo antitumor activity of T-DM1 was evaluated in three xenograft mouse models using cell lines with different levels of HER2 expression. HER2 expression in xenografts was assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), according to gastric cancer criteria. Cell cycle was assessed by flow cytometry after staining with propidium iodide. Apoptosis was assessed by caspase 3/7 activity measurement. Results: In the 17 BTC cell lines, HER2 expression was high in KMCH-1, Mz-ChA-1, and TGBC-18-TKB. Although cell growth inhibition of DM1 was similar in all cell lines (50% inhibitory concentration (IC50): 0.79–7.2 nM), that of T-DM1 was HER2-expression-dependent, with IC50 values in KMCH-1, Mz-ChA-1, and KKU-100 (which has low HER2 expression) of 0.031, 1.3, and 4.3 μg/mL, respectively. In KMCH-1 and Mz-ChA-1 xenograft models, T-DM1 treatment once every 3 weeks for 6 weeks showed significant dose-dependent antitumor activity, and tumor growth inhibition 21 days after first dose at 20 mg/kg T-DM1 was 108% (KMCH-1) and 75% (Mz-ChA-1), whereas there was no significant efficacy in KKU-100 xenograft model. Values for HER2 status (IHC score/FISH ratio of HER2 to CEP17) in KMCH-1, Mz-ChA-1, and KKU-100 tumor tissues were 3+/3.5, 2+/4.7, and 0/1.1, respectively. In KMCH-1, T-DM1 induced M phase arrest and apoptosis, as is reported in HER2-positive breast cancer cells. Conclusions: T-DM1 could be an effective therapy for HER2-overexpressed BTC.