FEZ1/LZTS1 is a tumor suppressor gene that is frequently altered in human cancers of different histotypes. We have reported previously that LZTS1 is downregulated in high-grade bladder cancer and that its restoration suppresses tumorigenicity in urothelial carcinoma cells. To further investigate the role of LZTS1 in the development of bladder cancer, we utilized heterozygous and nullizygous Lzts1 mice in a chemically induced carcinogenesis model. Fifty-eight mice consisting of 25 Lzts1(+/+), 17 Lzts1(+/-) and 16 Lzts1(-/-) were treated with N-butyl-N-(4-hydroxybutil) nitrosamine (BBN). Results showed that there was a significant increase in neoplastic lesions in the Lzts1(+/-) (82.3%) and Lzts1(-/-) (93.8%) versus Lzts1(+/+) (8.0%) mice after BBN treatment. No difference in cancer incidence between Lzts1(+/-) and Lzts1(-/-) was observed. Collectively, these findings indicate that loss of one or both LZTS1 alleles hampers the normal defenses of urothelial cells against carcinogens, favoring bladder cancer development. Therefore, LZTS1 may become an excellent target for gene therapy in advanced bladder carcinoma.