Background: BTCs have an aggressive tumor biology with limited treatment options. With a HER2-positivity rate of 5–20% in BTCs, case series and small clinical trials have shown signs of activity for HER2 blockade in these pts. T-DXd is an antibody-drug conjugate composed of a humanized monoclonal anti-HER2 antibody, a cleavable linker, and a topoisomerase I inhibitor. The HERB trial is an investigator-initiated, multicenter, single-arm phase 2 trial of T-DXd in pts with HER2-expressing BTCs. Methods: Centrally confirmed HER2-expressing (HER2-positive: IHC3+ or IHC2+/ISH+, and HER2-low-expressing [HER2-low]: IHC/ISH status of 0/+, 1+/-, 1+/+, or 2+/-) pts with BTCs who were refractory or intolerant to gemcitabine containing regimen received 5.4 mg/kg of T-DXd every 3 weeks. The primary endpoint was the confirmed objective response rate (ORR) in HER2-positive pts by independent central review. The sample size of 22 had 80% power with one-sided alpha error of 5%; threshold ORR, 15%; and expected ORR, 40%. The ORR, disease control rate (DCR), progression-free survival (PFS), overall survival (OS) in HER2-positive/-low pts, and incidence of treatment-emergent adverse events (TEAEs) were assessed as secondary endpoints. Results: A total of 32 pts, 24 with HER2-positive and 8 with HER2-low BTCs, received T-DXd. Twenty-two pts with HER2-positive, excluding 2 ineligible pts, were identified for primary efficacy analysis. Among the 22 pts, IHC3+ and IHC2+/ISH+ were 45.5% and 54.5%, primary sites: gallbladder/extrahepatic/intrahepatic/Vater were 11/6/3/2, median number of prior regimens was 2 (range, 1–4). The confirmed ORR in HER2-positive pts was 36.4% (8/22; 2 CR and 6 PR; 90% CI, 19.6–56.1), indicating statistically significant improvement in ORR (P = 0.01). The DCR, median (m) PFS, mOS were 81.8% (95% CI, 59.7–94.8), 4.4 months (mo) (95% CI, 2.8–8.3), 7.1 mo (95% CI, 4.7–14.6), respectively. In addition, encouraging efficacy were seen even in HER2-low pts; ORR, DCR, mPFS, and mOS were 12.5% (1/8; 1 PR; 95% CI, 0.3–52.7), 75.0% (95% CI, 34.9–96.8), 4.2 mo (95% CI, 1.3–6.2), and 8.9 mo (95% CI, 3.0–12.8), respectively. In the safety analysis set (n = 32), TEAEs of > = grade (G) 3 occurred in 81.3% (26/32); the common TEAEs were anemia (53.1%), neutropenia (31.3%), and leukopenia (31.3%). TEAEs leading to drug discontinuation occurred in 8 pts (25.0%). Eight pts (25.0%) had interstitial lung disease (ILD; G1/G2/G3/G5 were 3/1/2/2) not adjudicated by an independent committee. Conclusions: T-DXd showed promising activity in pts with HER2-expressing BTCs. Although the safety profile was generally consistent with other T-DXd studies, ILD, an important identified risk of T-DXd, requires more careful monitoring and intervention. These results support further exploration of T-DXd in this patient population. Clinical trial information: JMA-IIA00423.