Background: Human epidermal growth factor receptor 2 (HER2) overexpression is related to anti-HER2 therapy in many tumors. RC48-antibody-drug conjugate (ADC) has shown promising efficacy in patients with HER2-positive locally advanced or metastatic urothelial carcinoma (UC). The characteristic expression and scoring systems of HER2 are nonexistent in UC. We aimed to explore HER2 status and its correlation with the efficacy of HER2-targeting ADC therapy in UC.Methods: A total of 137 and 43 patients were enrolled in cohort 1 and cohort 2, respectively, from March 2009 to December 2018. The patients in cohort 2 were enrolled in a phase II study of RC48-ADC. UC samples were tested for HER2 status using immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). The 2018 ASCO/CAP HER2 scoring system was adopted and modified to score HER2 expression in UC.Results: The HER2-positive (IHC 2 + or 3 + ) rate was 24.1% (33/137). In HER2 IHC 2 + or 3 + patients, the HER2 gene amplification rate was 31% (13/42). The objective response rates (ORRs) in RC48-ADC-treated patients with IHC 3 + , IHC 2 + and FISH + , IHC 2 + and FISH-were 58.8%, 66.7% and 40%, respectively. The ORR showed a trend toward a better benefit for RC48-ADC therapy in patients with HER2 amplification than in those without amplification (61.5% vs. 44.8%, P = 0.059). The heterogeneity of HER2 expression in the primary tumor was 55.5% (15/27), and the ORR was not significantly different between patients with tumor heterogeneity and homogeneity.Conclusions: IHC testing should be performed to assess the HER2 status before the initiation of HER2-ADC therapy. There was a trend toward a better benefit for patients with HER2 amplification, and tumor heterogeneity did not influence the drug efficacy.