The FOXO4 transcription factor plays an important role in cell survival in response to oxidative stress. The regulation of FOXO4 is orchestrated by post-translational modifications including phosphorylation, acetylation, and ubiquitination. Here, we demonstrate that O-GlcNAcylation also contributes to the FOXO4-dependent oxidative stress response. We show that hydrogen peroxide treatment of HEK293 cells increases FOXO4 association with OGT, the enzyme that adds O-GlcNAc to proteins, causing FOXO4 O-GlcNAcylation and enhanced transcriptional activity under acute oxidative stress. O-GlcNAcylation is known to be protective for cells under stress conditions, including oxidative stress. Our data provide a mechanism of FOXO4 anti-oxidative protection through O-GlcNAcylation. Structured summary: MINT-7299700, MINT-7299716: Foxo4 (uniprotkb:Q9WVH3) physically interacts (MI:0915) with Ogt (uniprotkb:P56558) by anti tag coimmunoprecipitation (MI:0007). MINT-7299691: Ogt (uniprotkb:O15294) physically interacts (MI:0915) with Foxo4 (uniprotkb:P98177) by anti bait coimmunoprecipitation (MI:0006). © 2009 Federation of European Biochemical Societies.