Purpose: To examine the role of microRNAs (miRNAs), miR-331-5p, in gastric cancer (GC). Methods: The mRNA level of miR-331-5p and protein level of 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase-3 (PFKFB3) were determined using quantitative real-time reverse transcription- polymerase chain reaction (qRT-PCR) and western blotting, respectively. The cell viability and proliferation of the two GC cell lines (AGS and MKN45) were evaluated using Cell Counting Kit-8 (CCK- 8) and bromodeoxyuridine (BrdU) assays. Cell migration and invasion of AGS and MKN45 were evaluated using wound healing and invasion assays, respectively. Potential interactions between miR- 331-5p and PFKFB3 were assessed by luciferase activity assay, while the effects of the interactions on cell physiology and metabolism were investigated in cells overexpressing both miR-331-5p and PFKFB3. Results: MiR-331-5p overexpression inhibited cell proliferation, suppressed migration and invasion, and inhibited glycolysis in AGS and MKN45 cells. The mRNA for the glycolytic regulatory enzyme PFKFB3 was shown to be a direct target of miR-331-5p and modulated by miR-331-5p. In rescue experiments, PFKFB3 reversed the miR-331-5p-induced inhibition of proliferation, migration, invasion, and glycolysis in AGS cells. Conclusion: This work supports a role for miR-331-5p through the modulation of PFKFB3 activity in GC in vivo, thus providing insight into novel potential therapies for the treatment of GC. [ABSTRACT FROM AUTHOR]