Folate insufficiency can induce carcinogenesis by decreasing DNA methylation. It is well known that DNA hypomethylation is a common feature in a number of cancers. Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are enzymes that play central roles in the folate metabolic pathway. Two common polymorphisms in the MTHFR gene (C677T and A1298C) and one in the MS gene (A2756G) are associated with decreased enzymatic activity. In this work, we have conducted a case-control study to assess the role of these three polymorphisms in bladder cancer development in North Tunisia. For MS A2756G, gene and genotypic distributions differed significantly between cases and controls. Furthermore, individuals carrying at least one copy of the variant allele presented a 2.33 times increased risk of developing bladder cancer than their control group [P = 0.001, odds ratio (OR) = 2.33; 95% confidence interval (CI) 1.34-4.06]. Statistically significant odds ratios were also found in patients heterozygous for MTHFR A1298C, who have a 1.8-fold higher risk of developing bladder cancer (P = 0.03, OR = 1.86; CI 95% 1.04-3.33). While the isolated polymorphism C677T did not appear to influence bladder cancer susceptibility, results suggest that it might act with an additive contribution determined by variation at MTHFR A1298C. Identical cumulative effect was detected for the MTHFR A1298C and MS 2756 genotypes. Patients harboring at least one mutant allele for each of the three positions analyzed showed a 4.76-fold increased risk of developing bladder cancer in comparison to their reference group (P = 0.02, OR = 4.76; CI 95% 1.26-17.98).