Expression of microRNA-129-5p (miR-129-5p) has been reported to decrease in gastric cancer (GC). However, little information is available about how miR-129-5p affects cell migration and invasion of GC. Cancer samples and matched non-tumor adjacent tissues were obtained from patients with GC. Besides, peripheral blood samples were collected from both the patients and healthy volunteers. Expression of miR-129-5p was analyzed by real-time PCR (RT-PCR). After transfection with miR-129-5p mimics, miR-129-5p inhibitor, or negative controls in human GC cell line SGC-7901, cell viability, colony-formation ability, migration, and invasion assay were evaluated. Luciferase reporter assay, RT-PCR, and enzyme-linked immunosorbent assay (ELISA) were performed to explore whether interleukin-8 was a target of miR-129-5p. Further, small interfering RNA (siRNA) against IL-8 was transfected into cells, and then the effects of miR-129-5p inhibitor on migration and invasion were assessed. MiR-129-5p was down-regulated in both GC samples and blood samples compared to their matched non-tumor adjacent tissues and healthy volunteers (both P < 0.05). Compared to the control group, transfection with miR-129-5p inhibitor markedly increased the cell viability, colony-forming ability, and numbers of migrated and invaded cells. Luciferase reporter assay confirmed that IL-8 was a direct target of miR-129-5p, and IL-8 was negatively regulated by miR-129-5p. Cotransfection of miR-129-5p inhibitor with si-IL-8 reversed the effect of miR-129-5p inhibitor on the migration and invasion of the cells. MiR-129-5p and regulates migration and invasion of GC cells by targeting IL-8.