MicroRNAs (miRNAs) are identified as key regulators in cancer initiation, progression and metastasis including gastric cancer (GC). The aim of the study is to explore clinical significance and potential mechanism of miR-129-5p in GC development. In the study, our results found that miR-129-5p expression was significantly downregulated in GC tissues, compared with adjacent normal tissues using qRT-PCR analyses. Furthermore, lower miR-129-5p expression closely associated with tumor size and lymph node invasion and poor prognosis of GC patients. Using CCK8 assay, cell colony formation, transwell invasion assay, we demonstrated that miR-129-5p overexpression reduced cell proliferation, cell colony formation and cell invasion capacity in MKN45 (higher miR-129-5p expression) and SGC-7901 (lower miR-129-5p expression). However, downregulation of miR-129-5p had reverse effects on cell proliferation and invasion. Targeting association analysis, dual luciferase assay, qRT-PCR and western blot analysis results verified that miR-129-5p could target the 3′UTR of ADAM9 mRNA and regulated its protein expression. Furthermore, we confirmed that miR-129-5p suppressed cell proliferation and invasion ability through regulating ADAM9. In vivo, upregulation of miR-129-5p also inhibited tumor growth. Therefore, these results indicated that miR-129-5p functioned as a tumor suppressor in GC and may be a potential target of GC treatment.