The functional single nucleotide polymorphism (SNP) C3435T in exon 26 of the ABCB1 gene encoding the xenobiotic transporter P-glycoprotein (P-gp) may influence susceptibility to several diseases, as well as the clinical outcome of treatment with P-gp substrates. Exposure to environmental chemicals is thought to be involved in peptic ulcer pathogenesis and then later in stomach cancer development. About 80% of ulcers are associated with Helicobacter pylori infection, one of the risk factors of stomach cancer. P-gp-transported drugs are used in treatment of H. pylori. Therefore, a lack of effectiveness in eradication therapy can lead to chronic stomach inflammation and promote cancerogenesis. In this study, 196 patients with peptic ulcers divided into two groups with and without H. pylori infection and combined with 96 healthy controls were genotyped for the ABCB1 C3435T SNP. A trend towards higher incidence of the 3435TT genotype among peptic ulcer patients than in controls (p = 0.0983) was observed. Likewise, the 3435T allele was more frequent in groups suffering from peptic ulcers. The association was near to statistical significance (p = 0.0538). Between analyzed genotypes and H. pylori infection, statistically significant dependence was found (p = 0.0372). In addition, the CT genotype was associated with 1.56 times and the TT with 2.45 times higher prevalence of infection compared to the CC genotype. Asimilar association was present in a subgroup of peptic ulcer men (p = 0.0090). The isolated C3435T ABCB1 SNP is not a major factor for genetic susceptibility to peptic ulcer, but in a group of men who suffered from peptic ulcer, this polymorphism seemed to be a risk factor for H. pylori infection development.