Even though the dynamic modification of polypeptides by the monosaccharide, O-linked N-acetylglucosamine (O-GlcNAcylation) was discovered over 30 years ago, its physiological significance as a major nutrient sensor that regulates myriad cellular processes has only recently been more widely appreciated. O-GlcNAcylation, either on its own or by its interplay with other post-translational modifications, such as phosphorylation, ubiquitination, and others, modulates the activities of signaling proteins, regulates most components of the transcription machinery, affects cell cycle progression and regulates the targeting/turnover or functions of myriad other regulatory proteins, in response to nutrients. Acute increases in O-GlcNAcylation protect cells from stress-induced injury, while chronic deregulation of O-GlcNAc cycling contributes to the etiology of major human diseases of aging, such as diabetes, cancer, and neurodegeneration. Recent advances in tools to study O-GlcNAcylation at the individual site level and specific inhibitors of O-GlcNAc cycling have allowed more rapid progress toward elucidating the specific functions of O-GlcNAcylation in essential cellular processes.