Cancer cells exhibit increased uptake of glucose and glutamine, and rewire the metabolic flux toward anabolic pathways important for cell growth and proliferation. Understanding how this altered metabolism is regulated has recently emerged as an intense research focus in cancer biology. O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of serine and/or threonine residues of nuclear and cytosolic proteins. O-GlcNAcylation has been identified in numerous proteins that are involved in many important cellular functions, including transcription, translation, signal transduction, and stress responses. More recently, increasing evidence indicates that O-GlcNAcylation plays important roles in regulating cancer metabolic reprogramming by modifying key transcription factors, metabolic enzymes and major oncogenic signaling pathways. Thus, O-GlcNAcylation emerges as a novel regulatory mechanism linking altered metabolism to cancer pathogenesis.