A study has shown that miR-423-5p is highly expressed in proliferative diabetic retinopathy. However, the exact biological functions and mechanisms of miR-423-5p in diabetic retinopathy (DR) progression are currently unclear. This study aimed to investigate the role of miR-423-5p in DR and the underlying mechanism. Our data demonstrate that the expression of miR-423-5p is significantly increased in HG-induced RPE cells and DR patient plasma. Moreover, the overexpression of miR-423-5p exacerbates HG-induced apoptosis. Mechanistically, our results provide evidence that miR-423-5p directly targets TFF1. MiR-423-5p exerts its effect on HG-induced apoptosis in RPE cells through TFF1, and the NF-κB pathway is involved in the regulatory mechanism. Further analysis revealed that the transcription factor NFE2 regulates miR-423-5p promoter activity. In addition, NFE2 regulates the levels of TFF1 and NF-κB pathway-associated proteins by regulating the expression of miR-423-5p. The NFE2-miR-423-5p-TFF1 axis is a novel molecular mechanism and provides a new direction for the study and treatment of DR.