-linked -acetylglucosamine (-GlcNAc) modification, known as -GlcNAcylation, is a dynamic post-translational modification involving the addition of -acetylglucosamine to serine or threonine residues. It has emerged as a critical regulator in diabetic pathophysiology. This review summarizes current research on the role of -GlcNAcylation in hyperglycemia-induced cellular dysfunction, and focuses on vascular smooth muscle cells, renal cytoskeletal proteins, and diabetic complications in animal and human models. Studies reveal that hyperglycemia upregulates -GlcNAc transferase activity, disrupting the interplay between glycosylation and phosphorylation, thereby impairing signaling pathways and exacerbating vascular proliferation and renal cytoskeletal disorganization. Notable findings include the imbalance of -actin modifications in diabetic nephropathy, correlated with podocyte damage and glomerular abnormalities. By elucidating these mechanistic pathways, this review underscores the potential of -GlcNAcylation as a biomarker and a therapeutic target. Future research should focus on tissue-specific effects and pharmacological strategies that mitigate diabetes-induced complications while preserving normal cellular functions.