To evaluate the efficacy and safety of RC48-ADC, a novel humanized anti-HER2 antibody conjugated with monomethyl auristatin E (MMAE), in patients with HER2-positive locally advanced or metastatic urothelial carcinoma (mUC) refractory to standard therapies. This was a phase 2, open-label, multicenter, single-arm study of patients with HER2-positive (immunohistochemical status 3+ or 2+) locally advanced or metastatic UC who previously failed with at least one line systemic chemotherapy. The primary endpoint was the objective response rate (ORR) assessed by a blinded independent review committee (BIRC). The secondary endpoint included progression-free survival (PFS), disease control rate, duration of response, overall survival (OS), and safety. Forty-three patients were enrolled. The median follow-up was 20.3 months. The overall confirmed ORR as assessed by the BIRC was 51.2% (95% CI: 35.5%, 66.7%). Similar responses were observed in prespecified subgroups, such as those with liver metastasis and those previously treated with anti-PD-1/L1 therapies. The median PFS and OS were 6.9 months (95% CI: 5.6, 8.9) and 13.9 months (95% CI: 9.1, NE), respectively. The most common treatment-related adverse events (TRAEs) were hypoaesthesia (60.5%), alopecia (55.8%), and leukopenia (55.8%). Twenty-five (58%) patients experienced Grade 3 TRAEs, including hypoaesthesia (23.3%) and neutropenia (14.0%). No Grade 4 or Grade 5 TRAEs occurred. RC48-ADC demonstrated a promising efficacy with a manageable safety profile in HER2-positive locally advanced or metastatic UC patients who had failed at least one line of systemic chemotherapy. Copyright ©2020, American Association for Cancer Research.