: The previous studies have shown that the retinoic acid (RA) metabolism is closely related to the cancer stemness, but its role in bladder cancer development has not been fully investigated. : We conducted a comprehensive analysis of mutations, copy number variations and transcriptional changes of RA metabolism related genes in bladder cancer cells. We evaluated the activity of RA metabolism in tumor cells by using single cell transcriptome data and identified differentially expressed genes (DEGs) in cell subsets with high RA-metabolism score. We also investigated and verified the biological function of (one of RA metabolism related genes) . Additionally, we analyzed and verified the relationship between and tumor immune microenvironment by multiplex immunohistochemical (mIHC). : Comprehensive analysis indicates that the mutation rate of RA metabolism related genes in bladder cancer is about 20%, with significant gene amplification observed in and We identified a group of subsets with significantly increased RA metabolism activity in bladder cancer tumor epithelial cells and found that this subgroup was significantly associated with poor prognosis (p < 0.05). was identified as a potential therapeutic target. It was found that is significantly correlated with tumor stemness and differentiation. experiments confirmed that overexpression of can significantly enhance the proliferation and migration of tumor cells. These results suggest that CYP26B1 may be closely related to the remodeling of the tumor microenvironment and may become a potential therapeutic target for bladder cancer.