High glucose (HG)-induced apoptosis of retinal ganglion cells (RGCs) contributes to the pathogenesis of diabetic retinopathy, which is one of the most common and severe complications of diabetes mellitus. Accumulating evidence has documented that microRNAs (miRNAs) play an important role in the pathogenesis of diabetic retinopathy. However, the role of miRNAs in regulating HG-induced apoptosis of RGCs remains largely unknown. Various studies have suggested that miR-495 is an important regulator of cell apoptosis and survival. In this study, we aimed to investigate whether miR-495 is involved in regulating HG-induced apoptosis of RGCs and reveal its possible relevance in diabetic retinopathy. We found that miR-495 was significantly upregulated in HG-treated RGCs. Downregulation of miR-495 protected RGCs against HG-induced apoptosis, whereas overexpression of miR-495 had the opposite effect. Notably, Notch1 was identified as a target gene of miR-495, as miR-495 negatively regulated Notch1 expression and the Notch signaling pathway. Moreover, downregulation of miR-495 inhibited PTEN expression while promoting Akt activation. However, knockdown of Notch1 significantly abolished the protective effect of miR-495 inhibition against HG-induced apoptosis. Overall, our study suggests that downregulation of miR-495 alleviates HG-induced apoptosis of RGCs by targeting Notch1 to regulate PTEN/Akt signaling, which provides novel insights into understanding the pathogenesis of HG-induced apoptosis of RGCs.