NAT10 is a vital acetyltransferase and mainly localizes in nucleus. Currently, the oncogenic function of nuclear and membranous NAT10 are clear. However, whether NAT10 functions in mitochondrial remains unknown. Here, we found that NAT10 is also localized in mitochondrial and catalyze the acetylaion of multiple enzymes in mitochondrial. Therefore, the mitochondrial NAT10 is a vital therapeutic target for cancer.