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Background: Gastric Cancer (GC) accounts for high mortality, which seriously threatens people’s health. This study set out to probe into the effect and mechanism of miR-27b-3p on invasion and migration of GC. Methods: The miRNA sequence data of GC was acquired from The Cancer Genome Atlas (TCGA) database. The Differential Expression of miRNAs (DEMis) was acquired through R packages “edgeR” and “limma.” TargetScan, picTar, RNA22, PITA, and miRanda were performed to predict the target gene of miR-27b-3p. Western-blot and RTPCR were applied to detect the expression level of the selected candidate. Transwell assays evaluated the effect of miR-27b-3p and runt-related transcription factor 1 (RUNX1) on cell migration and invasion. The rescue assay was achieved by co-culture with mimics of miR-27b-3p and vector of RUNX1. The psiCHECK2 vector was used in luciferase report assay. Results: We found miR-27b-3p was down-regulated in GC and associated with GC patients' poor survival based on the TCGA data and bioinformatics analysis. Furthermore, RUNX1 was the target gene of miR-27b-3p, which was proved by luciferase report assay. miR-27b-3p and RUNX1 jointly participate in the regulation of Hippo pathway. The upregulated miR-27b-3p could inhibit Epithelial–Mesenchymal Transition (EMT) as well as invasion and migration. However, an overexpressed RUNX1 could weaken this phenomenon. Conclusion: MiR-27b-3p was down-regulated in GC, and it could regulate Hippo pathway and affect EMT by inhibiting RUNX1 expression.