Cumulatively, our results suggested that SIRT3 activation could regulate the acetylation status and stability of mito-COX-2 translocated to mitochondria, mediating the mito-COX-2/p-Drp1Ser616 interaction to promote the mitochondrial fission-driven functional outcomes and anti-tumor sensitivity of HCC cells in vitro and in vivo. This evidence concerns the gene SIRT3 and hepatocellular carcinoma.