Introduction: Recurrent urinary tract infections have been linked to increased risk of bladder cancer, suggesting a potential role of the urinary microbiome in bladder cancer pathogenesis.Objective: Compare the urinary microbiomes in mice with and without bladder. Methods:Longitudinal study of mice exposed to a dilute bladder-specific carcinogen (0.05% nbutyl-n-(4-hydroxybutyl) nitrosamine, BBN mice, n=10), and control mice (n=10). Urine was sampled monthly from individual mice for 4 months. Microbial DNA was extracted from the urine, and the V4 region of the 16S rRNA gene sequenced. Animals were sacrificed and their bladders harvested for histopathology. Bladder sections were graded by a blinded pathologist. The composition and diversity of the urinary microbiome were compared between the BBN and control mice. Metabolic pathway analysis was completed using PICRUST.Results: Bladder histology in the BBN group showed normal tissue with inflammation (BBNnormal, n=5), precancerous pathologies, (BBN-precancerous, n=3), and invasive cancer (BBNcancer, n=2). Alpha diversity did not differ between the mice exposed to BBN and the control mice at any timepoint. There were no differences in the urinary microbiomes between the BBN and control mice at baseline. At month 4, mice exposed to BBN had higher proportion of both Gardnerella and Bifidobacterium compared to control mice. There were no differences in proportions of specific bacteria between either the BBN-precancer or BBN-cancer and controls at month 4. However, the BBN-normal mice had higher proportions of Gardnerella, Haemophilus, Bifidobacterium, and Ureaplasma Actinobaculum, and lower proportions of Actinomyces, compared to control mice at month 4. Functional pathway analysis demonstrated increases in genes related to purine metabolism, phosphotransferase systems, peptidases, protein folding, and bacterial toxins in the BBN-mice compared to control mice at month 4.Conclusion: Mice exposed to 4 months of BBN, a bladder-specific carcinogen, have distinct urine microbial profiles compared to control mice.