The aim of the study is to detect the expression of FOXO1 mRNA and protein in samples from gastric cancer patients with Helicobacter pylori (H. pylori) infection, and to investigate the relationship between FOXO1 expression and miR-183 expression. Twenty-six gastric cancer patients with H. pylori infection and 26 gastric cancer patients without H. pylori infection were included into experimental group and control group, respectively. Tumor tissues and peripheral blood were collected from all subjects. QRT-PCR was used to determine the expression of miRNA and mRNA. Western blotting was carried out to measure protein expression. Dual luciferase reporter assay was used to identify direct interaction between miRNA and 3'-UTR of mRNA. Cell proliferation was examined by CCK-8 assay. FOXO1 mRNA and protein expression was down-regulated in gastric cancer patients, being possibly related to H. pylori infection. The expression of miR-183 in tumor tissues and serum from gastric cancer patients with H. pylori infection was elevated, and probably regulated the expression of FOXO1 by direct targeting. Stimulation by H. pylori up-regulated the expression of miR-183 in gastric cancer AGS cells, and reduced the levels of FOXO1 mRNA and protein. Inhibition of miR-183 elevated the expression of FOXO1 and suppressed the proliferation of AGS cells. The present study demonstrates that the expression of FOXO1 in tumor tissues and blood from gastric cancer patients with H. pylori infection is significantly down-regulated, and may be related to the up-regulation of miR-183. H. pylori may regulate FOXO1 expression through miR-183 to affect the pathological process of gastric cancer.©The Author(s) 2023. Open Access. This article is licensed under a Creative Commons CC-BY International License.