BackgroundNutrient limitations often lead to metabolic stress during cancer initiation and progression. To combat this stress, the enzyme heme oxygenase 1 (HMOX1, commonly known as HO-1) is thought to play a key role as an antioxidant. However, there is a discrepancy between the level of HO-1 mRNA and its protein, particularly in cells under stress. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) is a recently discovered cellular signaling mechanism that rivals phosphorylation in many proteins, including eukaryote translation initiation factors (eIFs). The mechanism by which eIF2α O-GlcNAcylation regulates translation of HO-1 during extracellular arginine shortage (ArgS) remains unclear.MethodsWe used mass spectrometry to study the relationship between O-GlcNAcylation and Arg availability in breast cancer BT-549 cells. We validated eIF2α O-GlcNAcylation through site-specific mutagenesis and azido sugar N-azidoacetylglucosamine-tetraacylated labeling. We then evaluated the effect of eIF2α O-GlcNAcylation on cell recovery, migration, accumulation of reactive oxygen species (ROS), and metabolic labeling during protein synthesis under different Arg conditions.ResultsOur research identified eIF2α, eIF2β, and eIF2γ, as key O-GlcNAcylation targets in the absence of Arg. We found that O-GlcNAcylation of eIF2α plays a crucial role in regulating antioxidant defense by suppressing the translation of the enzyme HO-1 during Arg limitation. Our study showed that O-GlcNAcylation of eIF2α at specific sites suppresses HO-1 translation despite high levels ofHMOX1transcription. We also found that eliminating eIF2α O-GlcNAcylation through site-specific mutagenesis improves cell recovery, migration, and reduces ROS accumulation by restoring HO-1 translation. However, the level of the metabolic stress effector ATF4 is not affected by eIF2α O-GlcNAcylation under these conditions.ConclusionsOverall, this study provides new insights into how ArgS fine-tunes the control of translation initiation and antioxidant defense through eIF2α O-GlcNAcylation, which has potential biological and clinical implications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-023-00924-4.