This study aimed to clarify the role of glutamine in atherosclerosis and its participating mechanism. Forty C57BL/6J mice were divided into wild control (wild Con), ApoE−/− control (ApoE−/− Con), glutamine + ApoE−/−control (Glut + ApoE−/− Con), ApoE−/− high fat diet (ApoE−/− HFD), and glutamine + ApoE−/− HFD (Glut + ApoE−/− HFD) groups. The degree of atherosclerosis, western blotting, and multiomics were detected at 18 weeks. An in vitro study was also performed. Glutamine treatment significantly decreased the degree of aortic atherosclerosis (p = 0.03). O‐GlcNAcylation (O‐GlcNAc), IL‐1β, IL‐1α, and pyruvate kinase M2 (PKM2) in the ApoE−/− HFD group were significantly higher than those in the ApoE−/− Con group (p < 0.05). These differences were attenuated by glutamine treatment (p < 0.05), and aggravated by O‐GlcNA transferase (OGT) overexpression in the in vitro study (p < 0.05). Multiomics showed that the ApoE−/− HFD group had higher levels of oxidative stress regulatory molecules (guanine deaminase [GUAD], xanthine dehydrogenase [XDH]), proinflammatory regulatory molecules (myristic acid and myristoleic acid), and stress granules regulatory molecules (caprin‐1 and deoxyribose‐phosphate aldolase [DERA]) (p < 0.05). These differences were attenuated by glutamine treatment (p < 0.05). We conclude that glutamine supplementation might alleviate atherosclerosis through downregulation of O‐GlcNAc, glycolysis, oxidative stress, and proinflammatory pathway.