Nivolumab monotherapy has been approved for the adjuvant treatment of adult patients with urothelial carcinoma who are at high risk of recurrence after undergoing radical resection of urothelial carcinoma based on results of the phase 3 CheckMate 274 trial, in which adjuvant nivolumab versus placebo demonstrated improvement in the primary endpoint of disease-free survival (DFS) in high-risk muscle-invasive urothelial carcinoma (MIUC). Identification of biomarkers associated with treatment outcomes can help refine patient selection, and inform on the immunobiology of disease. To assess the relevance of key biomarkers in the adjuvant MIUC setting, extensive exploratory analyses of tumor biomarkers, including associations with DFS, were performed. Differential gene expression and gene signature analysis found that immune-related genes and pathways, in particular a high interferon-γ signature, were predictive of improved DFS in nivolumab-treated patients. Positive predictive and prognostic associations, respectively, were found for CD4 gene expression and measures of CD8 T cell infiltration. A composite predictive model suggested that high tumor cell PD-L1 expression, high CD4 gene expression, high tumor mutational burden score, receipt of neoadjuvant cisplatin and low transforming growth factor-β gene signature score made the greatest contributions to predicting improved outcomes in nivolumab-treated patients. These results reinforce studies establishing the importance of tumor biomarkers of adaptive immunity in influencing response to PD-1-PD-L1 blockade, indicating the potential predictive rather than solely prognostic nature of such findings. ClinicalTrials.gov identifier: NCT02632409 .