Mammalian cells metabolize glucose primarily for energy production, biomass synthesis, and posttranslational glycosylation; and maintaining glucose metabolic homeostasis is essential for normal physiology of cells. Impaired glucose homeostasis leads to hyperglycemia, a hallmark of diabetes mellitus. Chronically increased glucose in diabetes mellitus promotes pathological changes accompanied by impaired cellular function and tissue damage, which facilitates the development of cardiovascular complications, the major cause of morbidity and mortality of patients with diabetes mellitus. Emerging roles of glucose metabolism via the hexosamine biosynthesis pathway (HBP) and increased protein modification via O-linked β-N-acetylglucosamine (O-GlcNAcylation) have been demonstrated in diabetes mellitus and implicated in the development of diabetic cardiovascular complications. This review will discuss the biological outcomes of the glucose metabolism via the hexosamine biogenesis pathway and protein O-GlcNAcylation in regulating cellular homeostasis, and highlight the regulations and contributions of elevated O-GlcNAcylation to the pathogenesis of diabetic cardiovascular disease.