Animal models of N‐butyl‐N‐(4‐hydroxy butyl) nitrosamine (BBN)‐induced urothelial carcinoma (UC), particularly bladder cancer (BC), have long been established. However, the rare incidence of BBN‐induced upper urinary tract UC (UTUC), which originates from the same urothelium as BC, remains elusive. The scarcity of animal models of UTUC has made it challenging to study the biology of UTUC. To address this problem, we tried to establish a novel mouse model of UTUC by treating multiple mice strains and sexes with BBN. The molecular consistency between the UTUC mouse model and human UTUC was confirmed using multi‐omics analyses, including whole‐exome, whole‐transcriptome, and spatial transcriptome sequencing. 16S ribosomal RNA metagenome sequencing, metabolome analysis, and dietary interventions were employed to assess changes in the gut microbiome, metabolome, and carcinogenesis of UTUC. Of all treated mice, only female BALB/c mice developed UTUC over BC. Multi‐omics analyses confirmed that the UTUC model reflected the molecular characteristics and heterogeneity of human UTUC with poor prognosis. Furthermore, the model exhibited increased Tnf‐related inflammatory gene expression in the upper urinary tract and a low relative abundance of Parabacteroides distasonis in the gut. Dietary intervention, mainly without alanine, led to P. distasonis upregulation and successfully prevented UTUC, as well as suppressed Tnf‐related inflammatory gene expression in the upper urinary tract despite the exposure to BBN. This is the first report to demonstrate a higher incidence of UTUC than BC in a non‐engineered mouse model using BBN. Overall, this model could serve as a useful tool for comprehensively investigating UTUC in future studies.
