Metabolite-coupled post-translation modifications (PTMs) plays a crucial role in the regulation of gene expression and protein functions. In this study, we present the identification and characterization of a novel protein modification, named lysine pyruvatylation (Kpy). We identified five pyruvatylation sites on core histones and 68 sites on non-histone proteins in mammal cells. The levels of Kpy can be increased by adding sodium pyruvate and are influenced by changes in the glycolytic pathway. Through a screening of potential enzymes, we identified SIRT3 as the “eraser” of Kpy, while HAT1 and p300 function as the “writers”. Collectively, this study uncovers a new type of PTM, pyruvatylation, which is derived from pyruvate, and reveals the key regulatory elements for the Kpy pathway, laying a foundation for studying its roles in diverse cellular processes.