Diabetes activates retinal matrix metalloproteinase-9 (MMP-9), and MMP-9 damages the mitochondria and augments capillary cell apoptosis. Our aim is to elucidate the mechanism responsible for MMP-9 activation. Histone modifications and recruitment of the nuclear transcriptional factor-κB (p65 subunit) at the MMP-9 promoter and the activity of lysine-specific demethylase 1 (LSD1) were measured in the retina from streptozotocin-induced diabetic rats. The role of LSD1 in MMP-9 activation was investigated in isolated retinal endothelial cells transfected with LSD1 small interfering RNA (siRNA). The results were confirmed in the retina from human donors with diabetic retinopathy. Diabetes decreased histone H3 dimethyl lysine 9 (H3K9me2) and increased acetyl H3K9 (Ac-H3K9) and p65 at the retinal MMP-9 promoter. LSD1 enzyme activity and its transcripts were elevated. LSD1 siRNA ameliorated the glucose-induced decrease in H3K9me2 and increase in p65 at the MMP-9 promoter, and prevented MMP-9 activation, mitochondrial damage, and cell apoptosis. Human donors with diabetic retinopathy had similar epigenetic changes at the MMP-9 promoter. Thus, activated LSD1 hypomethylates H3K9 at the MMP-9 promoter and this frees up that lysine 9 for acetylation. Increased Ac-H3K9 facilitates the recruitment of p65, resulting in MMP-9 activation and mitochondrial damage. Thus, the regulation of LSD1 by molecular or pharmacological means has the potential to retard the development of diabetic retinopathy.