Objective To investigate the effect of miR-125b on the aerobic glycolysis of osteosarcoma HOS cells and its underlying mechanism. Methods Real-time quantitative PCR was performed to detect the expression of miR-125b in HOSB normal human osteoblast cells and HOS osteosarcoma cells. The glucose uptake rate was assessed with 3H-2 deoxyglucose (3H-2DG) and lactate production was tested with the kits to observe the effect of miR-125b-mimics on the aerobic glycolysis of osteosarcoma HOS cells. Hexokinase-2 (HK2) protein was detected by Western blot analysis. Dual luciferase reporter gene assay was used to determine whether HK2 was the direct target of miR-125b. Results Compared with HOSB normal human osteoblast cells, the expression of miR-125b was significantly lower in HOS cells. The glucose uptake and lactate production were downregulated in HOS cells transfected with miR-125b-mimics. Aerobic glycolysis of HOS cells was markedly inhibited. Protein level of HK2 was significantly inhibited in HOS cells transfected with miR-125b-mimics. Luciferase assay indicated that HK2 was the direct target of miR-125b. Conclusion miR-125b is down-regulated in HOS cells, and it inhibits the aerobic glycolysis of HOS cells by directly regulating the expression of HK2.