PURPOSE: Platinum-based chemotherapy and immune checkpoint inhibitors are key components of systemic treatment for muscle-invasive and advanced urothelial cancer. The ideal integration of these two treatment modalities remains unclear as clinical trials have led to inconsistent results. Modulation of the tumor-immune microenvironment by chemotherapy is poorly characterized. We aimed to investigate this modulation, focusing on potential clinical implications for immune checkpoint inhibitor response. EXPERIMENTAL DESIGN: We assessed immune cell densities, spatial relations, and tumor/stromal components from 116 patients with urothelial bladder cancer (paired data for 95 patients) before and after platinum-based chemotherapy. RESULTS: Several published biomarkers for immunotherapy response changed upon chemotherapy treatment. The intratumoral CD8+ T-cell percentage increased after treatment and was associated with increased TNFα-via-NF-κB signaling. The percentage of PDL1+ immune cells was higher after chemotherapy. An increase in chemo-induced changes that potentially inhibit an antitumor immune response was also observed, including increased fibroblast-based TGFβ signaling and distances from immune cells to the nearest cancer cell. The latter two parameters correlated significantly in posttreatment samples, suggesting that TGFβ signaling in fibroblasts may play a role in spatially separating immune cells from cancer cells. We examined specific chemotherapy regimens and found that treatment with methotrexate, vinblastine, doxorubicin, and cisplatin was associated with an increase in the macrophage cell percentage. Gemcitabine-containing chemotherapy was associated with upregulation of fibroblast TGFβ signaling. CONCLUSIONS: The opposing effects of platinum-based chemotherapy on the immune cell composition and stromal context of the tumor-immune microenvironment may explain the inconsistent results of clinical trials investigating chemotherapy and immune checkpoint inhibitor combinations in bladder cancer.