The dysregulated energy metabolism of glioblastoma cells is a critical factor in tumor development and progression. Lysine acetylation, regulated by the mitochondrial enzyme SIRT3, is known to play a key role in the metabolic phenotype of glioblastoma. To better understand how SIRT3 regulates mitochondrial metabolism, we inhibited SIRT3 and examined the proteome and acetylome of two glioblastoma cell lines with different metabolic preferences. Our results indicate that protein synthesis machineries are regulated by lysine acetylation and play a role in the metabolic phenotype. We also identified new SIRT3 targets that have not been previously associated with specific functions, highlighting their potential as future targets for further study. These findings enhance our understanding of the complex regulation of energy metabolism in glioblastoma and provide potential targets for future therapies.