Methylating agents are involved in bladder carcinogenesis. O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes methyl group from O6-methylguanine and thus plays an important role in the etiology of cancer. We hypothesized that two MGMT polymorphisms in exon 3, C16195T (or MGMT L53L) and C16286T (or MGMT L84F) are associated with risk of bladder cancer. In a hospital-based case-control study of 167 patients with bladder cancer and 204 cancer-free controls frequency-matched by age, sex, smoking status, and alcohol use, we genotyped these two MGMT polymorphisms. We found that these two polymorphisms alone had a non-significant main effect on risk of bladder cancer. However, when these two polymorphisms were evaluated together, individuals with the combined genotypes or haplotypes with one or more variant alleles (i.e. the 16195T and 16286T alleles) had statistically significantly increased risk of bladder cancer (adjusted odd ratio [OR]=1.67, 95% confidence interval [CI], 1.01-2.77) compared with those with no variant allele. In the stratification analysis, the risk of bladder cancer was increased in a dose-response manner as the age increased (P(trend)=0.010), and the increased risk was more pronounced among old subjects (>65 years) (adjusted OR=2.51, 95% CI, 1.05-6.04), men (1.76, 1.00-3.10), and non-drinkers (1.91, 1.08-3.36). In conclusion, these two MGMT polymorphisms may jointly play a role, in the etiology of bladder cancer in southern Chinese population. Larger studies are warranted to validate our findings.