T regulatory cells (Tregs) and related-cytokines are effectively engaged in the process of tumor immune escape and functionally inhibit immune response against the tumor. This study aimed to investigate the association of Foxp3 gene single nucleotide polymorphism (SNP) (rs3761548) with serum IL-35, IL-10, and TGF-β levels in gastric adenocarcinoma (GA) patients. The blood samples were obtained from 150 GA patients and 166 control subjects. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was done to genotyping of Foxp3 gene polymorphism (rs3761548). The serum cytokines levels were measured using the ELISA method. According to genotyping, the AA, and AC genotypes and A allele demonstrated significantly greater risk of GA. Considering the Lauren classification, our results revealed a greater risk of GA progression in patients with AC + AA genotype compared to CC genotype. Moreover, significantly increased levels of IL-10, IL-35, and TGF-β were observed in GA patients compared to controls and also in diffuse-type compared to the intestinal type of GA patients. The IL-35, IL-10 concentrations in GA patients displayed significant differences between the participants with CC, AC and AA genotypes. Further analysis indicated the prognostic role of serum IL-35, IL-10, and TGF-β levels in GA patients. Our results confirmed that the Foxp3 polymorphism (rs3761548) could influence the predisposition to GA and the serum IL-10, IL-35, and TGF-β levels. Thus, this polymorphism might be involved in the GA progression through influencing Tregs function and the secretion of immunomodulatory cytokines.