Diabetic retinal disease (DR) is the main cause of visual disability and blindness in adults with diabetes mellitus. Currently, efficient prevention and treatment are still under investigation. MicroRNAs (miRNAs) are groups of short, non-coding RNAs that post-transcriptionally control their target genes' expression through complementary binding to the 3'UTR region. MiRNAs have been reported to play important roles in a variety of physiological and pathophysiological processes. However, the roles of miR-125b in DR are still unclear. In this study, we exposed human retinal pigment epithelial (RPE) cells to high glucose levels to mimic DR progression. Hyperglycemia induced RPE cell death in 1, 3 and 5 days. Meanwhile, we observed that miR-125b expressions were significantly downregulated by the high glucose treatments. We demonstrated elevated cellular glycolysis rates of RPE cells under hyperglycemia. The glycolysis key enzymes, GLUT1, Hexokinase 2 (HK2) and LDHA were upregulated by high glucose. Moreover, treatments of RPE cells with low-toxic dosages of the glycolysis inhibitor, 2-DG or Oxamate, rescued the high glucose-induced cell from death. We identified hexokinase 2 as a direct target of miR-125b in RPE cells by showing the binding of the miR-125b seed region to HK2 mRNA 3'UTR. Notably, we demonstrated that the overexpression of miR-125b significantly attenuated hyperglycemia-induced RPE cell death. This study reveals a new mechanism for miRNA-mediated cellular protection against RPE cell death, representing an effective DR-treatment approach.