Immunotherapy offers a second-line option for patients with metastatic urothelial carcinoma (mUC) who failed standard therapy, but the biomarkers for predicting response remain to be explored. This study aims to evaluate the safety, efficacy, and correlative biomarker of toripalimab in patients with previously treated mUC.Patients with mUC received toripalimab 3 mg/kg Q2W. Clinical response was assessed every 8 weeks by an independent review committee per RECIST v1.1. Tumor PD-L1 expression, tumor mutational burden (TMB), and other biomarkers were evaluated.Among the intention-to-treat population (n = 151), 85% of the patients experienced treatment-related adverse event (TRAE) and 20% experienced grade 3 and above TRAE. The objective response rate (ORR) was 26% with a disease control rate (DCR) of 45%. The median duration of response, progression-free survival (PFS) and overall survival (OS) were 19.7 months (95% CI: 13.9 to NE), 2.3 months (95% CI: 1.8 to 3.6) and 14.4 months (95% CI: 9.3 to 23.1), respectively. Both PD-L1+ and TMB-high (10 mutations/Mb as the cut off) patients had better ORR than PD-L1- patients (42% versus 17%, p = 0.002) and TMB low patients (48% versus 22%, p = 0.014), respectively. The TMB-high group also showed better PFS (12.9 versus 1.8 months, p < 0.001) and OS (not reached versus 10.0 months, p = 0.018) than the TMB-low group.Toripalimab has demonstrated encouraging clinical activity in the second-line treatment of mUC with a manageable safety profile. PD-L1 expression and TMB were two independent biomarkers in the study.Copyright ©2021, American Association for Cancer Research.