Glioblastoma is a central nervous system cancer type with a poor prognosis. One of its main features is the constant change of metabolism phenotype to support its development and progression. Mitochondria play a critical role in cellular metabolism. Acetylation on lysine residues of mitochondrial enzymes is a mechanism for regulating protein function. This modification negatively affects the mitochondrial proteome function and is counteracted by the enzyme sirtuin 3 (SIRT3). To better understand how SIRT3 regulates mitochondrial metabolism, we inhibited SIRT3 and examined by high-resolution mass spectrometry analysis the proteome and acetylome of two glioblastoma cell lines with different metabolic preferences. Our results indicate that protein synthesis machinery is regulated by lysine acetylation and plays a role in the metabolic phenotype. We also identified new SIRT3 targets not previously associated with specific functions, highlighting their potential as future targets for further study. This study shed light on the role of SIRT3 in mitochondria and its implication for cellular metabolism. We have generated knowledge at the proteome and acetylome levels on the dynamics between two glioblastoma cell lines with opposing metabolic phenotypes. Our results might guide in searching for biomarkers at both proteome and acetylome levels in glioblastoma.