BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has emerged as a new target for metastatic urothelial carcinomas (mUCs). Disitamab vedotin (DV), an anti-HER2 antibody-drug conjugate (ADC), demonstrates a promising efficacy in patients with HER2-positive mUC. However, the role of DV in HER2-negative and HER2-low mUCs remains unknown. METHODS: Patients with HER2-negative and HER2-low (immunohistochemistry [IHC] 0 or 1+) mUCs who received ≥1 line of systemic chemotherapy were included. Patients received 2 mg/kg DV intravenously once every 2 weeks. The primary endpoint was the objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. FINDINGS: Nineteen patients with UC were enrolled from September 2019 to December 2021. The median age was 64.0 years. 15 patients (79%) had visceral metastases. HER2 IHC 0 and 1+ were detected in 6 and 13 patients, respectively. As of September 30, 2022, the confirmed ORR was 31.6% (95% confidence interval [CI]: 12.6, 56.6), and the disease control rate was 94.7% (18/19). The median PFS and OS were 5.5 (95% CI: 3.9, 5.7) and 16.4 (6.8, 26.8) months, respectively. Common treatment related adverse events were mostly grade 1 or 2, including leukopenia (52.6%) and hypoesthesia (47.4%). CONCLUSIONS: This is the first exploratory trial demonstrating substantial anti-tumor activity and a manageable safety profile using a HER2-targeting agent in patients with HER2-low mUC. This study was registered at ClinicalTrials.gov (ClinicalTrials.gov: NCT04073602). FUNDING: The study was funded by the Natural Science Foundation of China, the Natural Science Foundation of Tibet Autonomous Region, the Beijing Xisike Clinical Oncology Research Foundation, and RemeGen, Ltd.