The expression profiles of nine bladder cancer cell lines were compared against a pool containing equal total RNA quantities of each of them. Lower expression of KiSS-1 was revealed in cells derived from the most advanced bladder tumors. When comparing 15 primary bladder tumors versus a pool of four bladder cancer cell lines, lower transcript levels of KiSS-1 were observed in the invasive bladder carcinomas as compared to superficial tumors. KiSS-1 expression ratios provided prognostic information. The expression pattern of KiSS-1 transcripts was analyzed using in situ hybridization in nine bladder cancer cells, paired normal urothelium and bladder tumor samples (n = 25), and tissue microarrays of bladder tumors (n = 173). We observed complete loss of KiSS-1 in all invasive tumors under study as compared to their respective normal urothelium. The expression of KiSS-1 was found to be significantly associated with histopathological stage. Patients with lower KiSS-1 expression showed a direct correlation with overall survival in a subset of bladder tumors whose follow-up was available (n = 69). We did not observe any significant differential KiSS-1 expression along cell cycle by sorting analysis. A potential tumor suppressor role in bladder cancer was revealed for KiSS-1. Moreover, it showed predictive value by identifying patients with poor outcome.