Although non-muscle invasive bladder cancer (NMIBC) is widely seen in men, most laboratory studies of new intravesical therapies to prevent NMIBC have been conducted on female animals. In addition, ozone (O-3) has been shown to be a beneficial agent as an intravesical application in the treatment of various disorders. In the current study, we evaluated the immunohistopathological and oxidative-antioxidative effects of intravesical O-3 treatment on n-methyl-n-nitrosourea (MNU)induced NMIBC. Male Wistar-Albino rats (n=51) were divided into four groups: sham (n=6), O-3 only (n=15), MNU only (n=15), and MNU+O3 (n=15). The MNU-only and MNU+O-3 groups received MNU, and the O-3-only group received saline every other week for 10 weeks. The MNU-only group received 1 ml saline in place of O-3 treatment, whereas the O-3-only and MNU+O-3 groups were treated with 1 ml 25 pg/nnl O-3 between the 7th and 12th weeks. Rat bladders were collected in the 15th week for immunohistopathology and oxidant-antioxidant quantitation. Oxidant-antioxidant parameters were determined by ELISA. Although all surviving rats in the MNU-only group had preneoplastic (4/11, 36.4%) or neoplastic changes (7/11, 63.6%), a completely normal urothelium was observed in 2 rats (2/12, 16.7%) in the MNU+O-3-group (P=0.478). More high-grade lesions were observed in the MNUonly group (4/11, 36.4%) than in the MNU+O-3 group (1/12, 8.3%) (P=0.120). All oxidant-antioxidant parameters significantly increased (P<0.05) in the O-3-only group compared with the sham group. However, only antioxidant superoxide dismutase was remarkably higher (178.9%, P=0.060) in the MNU+O-3 group compared with the MNU-only group. This is the first methodologically and pathologically well-described male rat orthotopic bladder carcinogenesis model with intravesical MNU and administration of O-3 in NMIBC.