Phosphoglycerate kinase (PGK1) catalyzes the reversible phosphotransfer reaction from 1, 3-bisphosphoglycerate (1, 3-BPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP. By controlling ATP and 3-PG levels, PGK1 plays an important role in coordinating energy production with biosynthesis and redox balance. In contrast to the extensive investigation of the transcriptional regulation of PGK1, little is known about its post-translational regulation. Here, we report that PGK1 is acetylated at lysine 220 (K220) and this acetylation inhibits PGK1 activity by disrupting the binding with its substrate, ADP. We have identified KAT9 and HDAC3 as the acetyltransferase and deacetylase, respectively, for PGK1. Moreover, we show there is molecular crosstalk between mTOR-mediated HDAC3 S424 phosphorylation and PGK1 K220 acetylation. Our study uncovers a previously unknown mechanism for the insulin and mTOR pathway in regulating glycolytic ATP production and cellular redox potential via HDAC3-mediated PGK1 deacetylation.