<p>E3 ubiquitin ligases are acknowledged as the principal catalysts in the ubiquitination process due to their capacity to identify, bind and recruit specific substrates for modification. However, knowledge about the expression patterns of E3 ligases and their contribution to the tumor heterogeneity of bladder cancer (BLCA) is still lacking. Here, we delineated two distinct subcategories of BLCA utilizing consensus clustering of variable expression patterns of E3 ligases from the TCGA database, outlining the functional characteristics and immune profiles of these subclusters. Crucially, these clusters offered valuable perspectives on the tumor immune microenvironment (TIME) and tumor response to immunotherapy. Additionally, we established and validated an E3 ligase-related prognostic model predicated on genes associated with E3 ligases, which robustly foretold the prognosis, TIME, and the efficacy of immunotherapy in BLCA patients. Besides, we systematically interrogated the correlation between the IC50 values of commonly used antitumor drugs and the E3 ligase-related risk score and expression levels of prognostic genes. Notably, we identified and validated that EMP1 inhibition synergized with the antitumor effects of oxaliplatin in T24 and 5637 BLCA cell lines. Furthermore, knockdown of SLC26A8, an E3 ligase-related prognostic gene, significantly promoted tumor progression in BLCA. In summary, we introduced an innovative E3 ligase-based classification framework and prognostic model for BLCA, presenting a potent and auspicious prognostic and immunotherapeutic benefit predictor for individual BLCA patients.</p>