Glycolysis is a catabolic process of glucose hydrolysis needed for energy and biosynthetic intermediates, whereas gluconeogenesis is a glucose production process important for maintaining blood glucose levels during starvation. Although they share many enzymes, these two processes are not simply the reverse of each other and are instead reciprocally regulated. Two key enzymes that regulate irreversible steps in these two processes are pyruvate kinase (PK) and phosphoenolpyruvate carboxy kinase (PEPCK), which catalyze the last and first step of glycolysis and gluconeogenesis, respectively, and are both regulated by lysine acetylation. Acetylation at Lys305 of the PKM (muscle form of PK) decreases its activity and also targets it for chaperone-mediated autophagy and subsequent lysosome degradation. Acetylation of PEPCK, on the other hand, targets it for ubiquitylation by the HECT E3 ligase, UBR5/EDD1, and subsequent proteasomal degradation. These studies established a model in which acetylation regulates metabolic enzymes via different mechanisms and also revealed cross talk between acetylation and ubiquitination. Given that most metabolic enzymes are acetylated, we propose that acetylation is a major posttranslational modifier that regulates cellular metabolism.