Folates are the common sources of DNA synthesis and methylation. Cigarette smoking and genetic susceptibility of folate enzymes are two suspected factors most closely associated with bladder cancer development. This study sought to determine the effect of smoking and genetic polymorphisms in folate metabolizing enzymes on the histological stage and grade of bladder tumors in Tunisian patients. A total of 130 patients with urothelial cell carcinomas were examined with respect to smoking status, MTHFR (5,10-methylenetetrahydrofolate reductase), MTR (methionine synthase), MTRR (methionine synthase reductase) and TYMS (thymidylate synthase) genotypes distribution. Our data have reported that tobacco, MTHFR, MTR and MTRR genotypes were not associated with bladder tumor stage. Only TYMS 3R*G/3R*C genotype was associated with increased risk of developing invasive tumors compared to reference group (RR=1.74; 95% CI: 0.97-3.12). When we studied the superficial bladder tumor group, we have shown a significant statistical differences for the TYMS 3R*G/2R genotype. This genotype presented a 1.68-fold increased risk of developing high grade tumors compared to reference group (RR=1.68; 95% CI: 1.12-2.54). Moreover, we have shown that patients having at least one copy of 2R allele were at 4.23-fold increased risk for developing high grade tumors compared to reference group (P=0.022).