KISS1 is a metastasis suppressor gene that is lost in several malignancies, including bladder cancer. We tested the epigenetic silencing hypothesis and evaluated the biological influence of KISS1 methylation on its expression and clinical relevance in bladder cancer. KISS1 hypermethylation was frequent in bladder cancer cells analyzed by methylation-specific PCR and bisulfite sequencing and was associated with low gene expression, being restored in vitro by demethylating azacytidine. Hypermethylation was also frequently observed in a large series of bladder tumors (83.1%, n = 804). KISS1 methylation was associated with increasing stage ( P = 0.001) and tumor grade ( P = 0.010). KISS1 methylation was associated with low KISS1 transcript expression by quantitative RT-PCR ( P = 0.037). KISS1 transcript expression was also associated with histopathological tumor stage ( P < 0.0005). Low transcript expression alone ( P = 0.003) or combined with methylation ( P = 0.019) was associated with poor disease-specific survival ( n = 205). KISS1 transcript expression remained an independent prognosticator in multivariate analyses ( P = 0.017). KISS1 hypermethylation was identified in bladder cancer, providing a potential mechanistic explanation (epigenetic silencing) for the observed loss of KISS1 in uroepithelial malignancies. Associations of KISS1 methylation and its expression with histopathological variables and poor survival suggest the utility of incorporating KISS1 measurement using paraffin-embedded material for tumor stratification and clinical outcome prognosis of patients with uroepithelial neoplasias.