The ubiquitin-specific protease 16 (USP16), initially identified as a histone H2A-specific deubiquitinase. However, whether endogenous USP16 localizes to mitochondria and, if so, how it regulates mitochondrial function remain unclear. Therefore, this study focused on USP16, systematically investigating its localization, function, and regulatory mechanisms in mitochondria through various experimental approaches. Methods： First, we systematically investigated the subcellular localization of USP16 using immunofluorescence (IF), subcellular fractionation, and immunoblotting analysis. To further explore the function of USP16 in mitochondria, we conducted USP16 rescue experiments in USP16 knockout (KO) HeLa cells and performed mitochondrial function assays. To uncover the specific mechanism of USP16 in mitochondria, we successfully identified the key substrate of USP16—the translocase of outer mitochondrial membrane 40 (TOM40)—using multi-omics mass spectrometry analysis, co-immunoprecipitation, and immunofluorescence experiments. To further validate the role of TOM40 in USP16-regulated mitochondrial function, we overexpressed TOM40 in USP16 KO cells and then performed mitochondrial functional assays to confirm whether TOM40 is the critical downstream target through which USP16 regulates mitochondrial function and maintains metabolic homeostasis. Results： We demonstrate that USP16 interacts with and regulate the stability TOM40, the core pore-forming subunit of the translocase complex in the outer mitochondrial membrane (TOM). Specifically, USP16 deubiquitinates K48-linked ubiquitin chains from lysine 175, 184, and 309 of TOM40. Knockout of USP16 disrupts mitochondrial homeostasis, resulting in impaired membrane potential, elevates reactive oxygen species, reduces ATP synthesis, and alters oxygen consumption rates. Expression of FLAG-tagged USP16 restores TOM40 protein levels and rescues mitochondrial function. The essential role of TOM40 in mitochondrial homeostasis is further supported by partial restoration of mitochondrial functions upon TOM40 re-expression in USP16 KO cells. Conclusion: These findings identify USP16 as a novel regulator of mitochondrial function and establish TOM40 as an essential target of USP16 in mitochondria, providing valuable insights into the mechanisms by which USP16, acting as a dual-function deubiquitinase, supports mitochondrial integrity and function.