The mitochondria play a crucial role in metabolism and energy production by generating adenosine triphosphate (ATP) through oxidative phosphorylation. Additionally, they are involved in maintaining intracellular calcium levels, facilitate nuclear communication, detoxify reactive oxygen species (ROS), and regulate apoptosis. While not all protein regulation pathways are fully understood, mitochondrial protein acetylation is known to have an inhibitory effect, and the SIRT3 deacetylase plays a key role in this process, particularly in relation to cancer development. Our study on lung cancer cells (A549) and breast cancer cells (MCF7) revealed significant regulation through the processes of protein acetylation and deacetylation, with distinct effects from SIRT3 inhibition observed in each cell type. In A549 cells, inhibiting SIRT3 appears to impede cancer progression. In contrast, in MCF7 cells, SIRT3 activates unfavorable cancer-related pathways, suggesting that SIRT3 may have an oncogenic role in A549 and a suppressive function in MCF7. These findings highlight SIRT3 as a promising therapeutic target in cancer treatment.