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APOBEC3B enzymes are endogenous carcinogenic mutagens. Metastatic urothelial carcinomas often harbor APOBEC3B-mediated mutations in which tCw to T or G substitution occurs. Here, we evaluated patient survival and CD8+ T-cell density according to APOBEC3B expression in patients with metastatic urothelial carcinoma who underwent cytotoxic chemotherapy. We performed a retrospective study on 94 patients with urothelial carcinoma who were treated with first line palliative chemotherapy. APOBEC3B expression and CD8+/CD3+ ratio of tumor-infiltrating lymphocytes were evaluated using immunohistochemistry. Kaplan–Meier survival curves were generated and the log-rank test was employed. The association between APOBEC3B expression and tumor-infiltrating lymphocytes was analyzed using Pearson’s chi-squared test. High APOBEC3B expression was detected in 71 of the 94 patients (75.5%). The median overall survival was longer in patients with high APOBEC3B expression (15 months) than in those with low expression (p = 0.045). The hazard ratio obtained based on the Cox regression analysis was 0.292 (95% confidence interval 0.118–0.723, p = 0.008). APOBEC3B expression was associated with the CD8+/CD3+ ratio (2.914, 95% confidence interval 1.030–8.249, p = 0.039). Collectively, APOBEC3B expression was an independent prognostic factor in patients with metastatic urothelial carcinoma treated with platinum-based chemotherapy. Tumor-infiltrating cytotoxic T cells were associated with APOBEC3B expression.