This study examined the promoter methylation of APO-1/CD95 (Fas) gene in bladder urothelial carcinoma and analyzed the relationship between the Fas promoter methylation and the biological behavior of bladder cancer. Promoter methylation of Fas gene was detected by methylation-specific PCR (MSP) in 4 bladder cancer cell lines, 50 human bladder urothelial carcinoma samples and l0 normal bladder tissue samples. Correlation of the aberrant methylation of Fas promoter with the clinicopathological parameters was statistically analyzed. The results showed that Fas was down-regulated at both mRNA and protein level in bladder cancer cell lines and tissue samples of bladder urothelial carcinoma. The positive rate of Fas protein expression in bladder urothelial carcinoma was 34.0% (17/50), significantly lower than that in normal bladder tissues (70.0%, 7/10) (P<0.01). Fas promoter methylation was detected, and the positive rate of Fas promoter methylation in bladder urothelial carcinoma was 42.0% (21/50), which was obviously higher than that in normal bladder tissues (0.0%, 0/10) (P<0.01). The aberrant methylation of Fas promoter was reversely correlated with Fas protein expression (P<0.05). Furthermore, the positive rates of Fas promoter methylation in high-grade and low-grade bladder urothelial carcinoma were 73.3% (11/15) and 34.2% (12/35), respectively, with significant difference shown (P<0.05). No statistical significance was found in the Fas promoter methylation among different clinical stages of bladder cancer. It was concluded that Fas promoter hypermethylation plays an important role in the pathogenesis of bladder urothelial carcinoma and may serve as a prognostic indicator of bladder urothelial carcinoma.