Ferroptosis of the retinal pigment epithelial (RPE) cells leads to retinal neuron injury and even visual loss. Our study aims to investigate the role of the SET domain with lysine methyltransferase 7/9 (SET7/9) in regulating high glucose (HG)-induced ferroptosis in RPE cells. The cell model was established by HG treatment. The levels of SET7/9 and Sirtuin 6 (SIRT6) were inhibited and Runt-related transcription factor 1 (RUNX1) was overexpressed through cell transfection, and then their levels in ARPE-19 cells were detected. Cell viability and apoptosis was detected. The levels of reactive oxygen species, malondialdehyde, glutathione, ferrous ion, glutathione peroxidase 4, and acyl-CoA synthetase long-chain family member 4 were detected. SET7/9 and trimethylation of histone H3 at lysine 4 (H3K4me3) levels in the RUNX1 promoter region and RUNX1 level in the SIRT6 promoter region were measured. The relationship between RUNX1 and SIRT6 was verified. SET7/9 and RUNX1 were highly expressed while SIRT6 was poorly expressed in HG-induced ARPE-19 cells. SET7/9 inhibition increased cell viability and inhibited cell apoptosis and ferroptosis. Mechanistically, SET7/9 increased H3K4me3 on the RUNX1 promoter to promote RUNX1, and RUNX1 repressed SIRT6 expression. Overexpression of RUNX1 or silencing SIRT6 partially reversed the inhibitory effect of SET7/9 silencing on HG-induced ferroptosis. In conclusion, SET7/9 promoted ferroptosis of RPE cells through the SIRT6/RUNX1 pathway.