Immune checkpoint blockade is a promising therapeutic strategy against various human malignancies. MicroRNAs (miRNAs/miRs) regulate gene expression, by repressing mRNA translation or promoting its degradation. The aim of the current study was to investigate the role and molecular mechanisms of miR‑140 in Helicobacter pylori (Hp)‑associated gastric cancer, and to examine its relationship with immune function in gastric cancer. Gastritis tissue samples from gastritis patients, and gastric cancer tissue samples from gastric cancer patients were collected for miR‑140 expression detection. miR‑140 expression was detected using reverse transcription‑quantitative polymerase chain reaction, and protein expression was measured by western blotting. TargetScan and dual luciferase reporter assays were used to reveal the association between miR‑140 and programmed cell death‑ligand 1 (PD‑L1). BGC823 cell proliferation was detected by MTT assays. Ex vivo immune analysis by flow cytometry and ELISA were used to analyze immune function. It was demonstrated that miR‑140 expression was significantly reduced in Hp‑positive gastric cancer. PD‑L1 was confirmed as a direct target of miR‑140 in gastric cancer cells. In addition, PD‑L1 expression was significantly increased in Hp‑positive gastric cancer. Overexpression of miR‑140 significantly suppressed gastric cancer cell proliferation through regulating PD‑L1 expression. In vivo experiments also revealed that miR‑140 markedly repressed tumor growth in the C57BL/6 mice. Furthermore, it was determined that the tumor‑suppressive role of miR‑140 in gastric cancer was associated with increased cytotoxic CD8+ T cell and reduced myeloid‑derived suppressive and regulatory T cell infiltration. miR‑140 significantly prevented mammalian target of rapamycin signaling in gastric cancer cells. Notably, these miR‑140 overexpression‑induced alterations were inhibited by PD‑L1 plasmid. These findings indicated that miR‑140 exerted an anti‑gastric cancer effect by targeting immune checkpoint molecule PD‑L1. Thus, miR‑140 may be a promising and novel immunotherapeutic target for gastric cancer treatment.