miR-613 has been demonstrated to play critical roles in tumorigenesis and progression of a various type of cancers. However, its role and expression significance remain unclear in gastric cancer (GC). We detected the expression of miR-613 in 176 paired GC tissues and adjacent normal tissues, and found that miR-613 was significantly downregulated in GC tissues and its downregulation was correlated with T stage, lymph node invasion and advanced AJCC stages. Moreover, miR-613 expression could be an independent prognostic factor of GC. Biological function analysis indicated that miR-613 inhibited cell proliferation and invasion. Further analysis suggested that miR-613 inhibited Warburg effect of GC cells. Mechanically, we identified that miR-613 could directly bind to the 3'UTR of PFKFB2, thereby suppressing the expression of PFKFB2, which in turn, regulating glycolysis metabolism and cell growth. In conclusion, miR-613 served as a tumor suppressor by targeting PFKFB2, indicating that detecting miR-613 and modulation of miR-613 expression could be potential marker and clinical approach in GC patients.