PURPOSE: Epigenetic deregulation is deeply implicated in the pathogenesis of bladder cancer (BC). KDM6A (Lysine (K)-specific demethylase 6A) is a histone modifier frequently mutated in BC. However, the molecular mechanisms of how KDM6A deficiency contributes to BC development remains largely unknown. We hypothesized that clarification of the pathogenic mechanisms underlying Kdm6a-mutated BC can help in designing new anti-cancer therapies. EXPERIMENTAL DESIGN: We generated mice lacking Kdm6a in the urothelium and crossed them with mice heterozygous for p53, whose mutation/deletion significantly overlaps with the Kdm6a mutation in muscle-invasive BC (MIBC). In addition, BBN (N-butyl-N-(4-hydroxybutyl) nitrosamine), a cigarette smoke-like mutagen, was used as a tumor-promoting agent. Isolated urothelia were subjected to phenotypical, pathological, molecular and cellular analyses. The clinical relevance of our findings was further analyzed using genomic and clinical data of patients with MIBC. RESULTS: We found that Kdm6a deficiency activated cytokine and chemokine pathways, promoted M2 macrophage polarization, increased cancer stem cells and caused BC in cooperation with p53 haploinsufficiency. We also found that BBN treatment significantly enhanced the expression of proinflammatory molecules and accelerated disease development. Human BC samples with decreased Kdm6a expression also showed activated proinflammatory pathways. Notably, dual inhibition of interleukin-6 and chemokine (C-C motif) ligand 2, upregulated in response to Kdm6a deficiency, efficiently suppressed Kdm6a-deficient BC cell growth. CONCLUSIONS: Our findings provide insights into multistep carcinogenic processes of BC and suggest molecular targeted therapeutic approaches for BC patients with Kdm6a dysfunction.