To maintain homeostasis under variable nutrient conditions, cells rapidly and robustly respond to fluctuations through adaptable signaling networks. Evidence suggests that the O-linked N-acetylglucosamine (O-GlcNAc) posttranslational modification of serine and threonine residues functions as a critical regulator of intracellular signaling cascades in response to nutrient changes. O-GlcNAc is a highly regulated, reversible modification poised to integrate metabolic signals and acts to influence many cellular processes, including cellular signaling, protein stability, and transcription. This review describes the role O-GlcNAc plays in governing both integrated cellular processes and the activity of individual proteins in response to nutrient levels. Moreover, we discuss the ways in which cellular changes in O-GlcNAc status may be linked to chronic diseases such as type 2 diabetes, neurodegeneration, and cancers, providing a unique window through which to identify and treat disease conditions.