Deregulated expression of chemokines in tumor microenvironment contributes to tumor metastasis by targeting distinct cells. Epithelial-derived neutrophil-activating peptide-78 (ENA78/CXCL5) is upregulated in many cancers and involved in tumor progression. The role and underlying mechanism of CXCL5 in gastric cancer (GC) metastasis remain unclear. In this study, we reported that the expression of CXCL5 was elevated in tumor tissues and positively associated with lymphatic metastasis and tumor differentiation. Stimulation by recombinant human CXCL5 (rhCXCL5) induced epithelial-mesenchymal transition (EMT) in GC cells through the activation of ERK pathway, which enhanced their migration and invasion abilities. The culture supernatant from tumor tissues also enhanced the migration and invasion abilities of GC cells, however, this effect was reversed by pre-treatment with CXCL5 neutralizing antibody. Further studies showed that rhCXCL5 could induce the expression of IL-6 and IL-23 in neutrophils through the activation of ERK and p38 signaling pathways, which in turn facilitated GC cell migration and invasion. The culture supernatant from tumor tissues showed similar effects on neutrophils in a CXCL5-dependent manner. Blockade of IL-6 and IL-23 with neutralizing antibodies reversed the induction of EMT and the increased migration and invasion abilities in GC cells by CXCL5-activated neutrophils. Moreover, CXCL5 activated neutrophils could promote gastric cancer metastasis in vivo. Taken together, our results indicate that CXCL5 acts on gastric cancer cells to induce EMT and mediates pro-tumor activation of neutrophils, which synergistically promotes the metastatic ability of GC cells.