Polymorphisms in the gene encoding the metabolic enzyme cytochrome P450 1A1 (CYP1A1) might contribute to the variability in individual susceptibility to gallbladder cancer (GBC). This study comprised 142 consecutive cases of proven GBC and 171 healthy participants of similar ethnicity. CYP1A1 6235 T/C transition was analyzed using polymerase chain reaction-restriction fragment length polymorphism. The CC genotype of CYP1A1 was significantly associated with a risk of GBC [odds ratio (OR) 2.3, 95% confidence interval (CI)=1.1-4.5, P=0.026], and the age- and sex-adjusted OR was 2.0. The CYP1A1 C allele frequency was also higher in GBC patients (OR 1.4, 95% CI=1.0-2.0, P=0.039). After sex stratification, the TC genotype and C allele were significantly higher in men and conferred a risk for GBC (OR 4.2, 95% CI=1.8-9.7, P=0.001; OR 2.2, CI=1.3-3.7, P=0.005). In addition, the TC genotype and C allele frequencies were significantly higher in GBC patients without associated gallstones (OR 2.4, 95% CI=1.2-4.7, P=0.011; OR 1.6, CI=1.0-2.4, P=0.023). The usage of tobacco (smoking or nonsmoking) by GBC patients showed a significant increase in cancer risk with the TC genotype (OR 4.1, 95% CI=1.3-11.9, P=0.012). It seems that the higher risk of the TC genotype in men may be related to tobacco usage.