Lysine lactylation (Kla) has recently been reported to participate in regulating transcription in human cells. However, the characterization, regulatory mechanism and functional consequence of Kla in prokaryotes remain unclear. Here, we report that YiaC functions as a lysine lactylase and that CobB serves as a lysine delactylase in the regulation of metabolism. We demonstrate that YiaC catalyzes the addition of Kla, while CobB erases this PTM both in vitro and intracellularly. Moreover, we show that YdiF can catalyze the formation of a lactyl-coenzyme A, which donates lactyl group for Kla. Quantitative proteomic analysis further reveals 446 endogenous Kla sites targeted by CobB and 79 candidates targeted by YiaC in Escherichia coli (E. coli). Furthermore, we present that Kla can influence the functions of metabolic enzymes. Interestingly, we demonstrate that CobB can specifically modulate the activity of PykF by regulating K382la, promoting glycolysis and bacterial growth. Our study identifies the regulatory enzymes and functional network of Kla and reveals a Kla-mediated molecular mechanism catalyzed by CobB for glycolysis regulation in E. coli.