Acetyl-CoA represents a key node in metabolism due to its intersection with many metabolic pathways and transformations. Emerging evidence reveals that cells monitor the levels of acetyl-CoA as a key indicator of their metabolic state, through distinctive protein acetylation modifications dependent on this metabolite. We offer the following conceptual model for understanding the role of this sentinel metabolite in metabolic regulation. High nucleocytosolic acetyl-CoA amounts are a signature of a 'growth' or 'fed' state and promote its utilization for lipid synthesis and histone acetylation. In contrast, under 'survival' or 'fasted' states, acetyl-CoA is preferentially directed into the mitochondria to promote mitochondrial-dependent activities such as the synthesis of ATP and ketone bodies. Fluctuations in acetyl-CoA within these subcellular compartments enable the substrate-level regulation of acetylation modifications, but also necessitate the function of sirtuin deacetylases to catalyze removal of spontaneous modifications that might be unintended. Thus, understanding the sources, fates, and consequences of acetyl-CoA as a carrier of two-carbon units has started to reveal its underappreciated but profound influence on the regulation of numerous life processes.