Lysine lactylation (Kla), a newly identified epigenetic mark triggered by lactate during glycolysis, including the Warburg effect, marks a pivotal juncture between metabolic pathways and gene regulation. The discovery of enzymes such as p300 and HDAC1/3 has been pivotal in deciphering the regulatory dynamics of Kla, though questions about additional regulatory enzymes, their specific Kla substrates, and the underlying functional mechanisms persist. Our investigation bridges these knowledge gaps by identifying SIRT1 and SIRT3 as key "erasers" of Kla, providing insights into their selective regulatory impact on both histone and non-histone proteins. Through a quantitative proteomic analysis in wildtype, SIRT1 knockout, and SIRT3 knockout HepG2 cells, we delineated a comprehensive landscape of Kla and lysine acetylation (Kac) sites. The results demonstrate a distinct specificity in the substrates modified by SIRT1 and SIRT3, underscoring their differentiated roles in cellular signaling pathways. Particularly, we highlight the role of specific Kla modifications, such as those on the M2 splice isoform of pyruvate kinase (PKM2), in modulating metabolic pathways and cell proliferation, thereby expanding the recognized implications of Kla beyond its epigenetic roles. Therefore, this study paves the way for deeper understanding of the functional phenotypes and mechanisms of Kla, offering new insights into its broader biological significance.