BACKGROUND & AIMSMetabolic dysfunction-associated steatohepatitis (MASH) is a common chronic liver disease worldwide. No effective pharmacological therapies for MASH have been developed; to develop such promising drugs, the underlying mechanisms regulating MASH need to be elucidated. Here, we aimed to determine the role of OTUD5 in MASH progression and identify a specific mechanism. METHODSThe expression levels of OTUD subfamily under palmitic acid/oleic acid (PAOA) stimulation were screened. OTUD5 expression was assessed in human liver tissues without steatosis, those with simple steatosis, and those with MASH. MASH models were developed in hepatocyte-specific Otud5-knockout mice that were fed high-fat high-cholesterol (HFHC) and high-fat high-cholesterol plus high-fructose/sucrose (HFF) diet for 16 weeks. RESULTSThe expression of OTUD5 was downregulated in fatty liver and was negatively related to the progression of MASH. Lipid accumulation and inflammation were exacerbated by Otud5 knockdown but attenuated by Otud5 overexpression under PAOA treatment. Hepatocyte-specific Otud5 deletion markedly exacerbated steatosis, inflammation, and fibrosis in the livers of two MASH mouse models. We identified voltage-dependent anion channel 2 (VDAC2) as an OTUD5-interacting partner; OTUD5 cleaved the K48-linked polyubiquitin chains from VDAC2, and it inhibited subsequent proteasomal degradation. The anabolic effects of OTUD5 knockdown on PAOA-induced lipid accumulation were effectively reversed by VDAC2 overexpression in primary hepatocytes. Metabolomic results revealed that VDAC2 is required for OTUD5-mediated protection against hepatic steatosis by maintaining mitochondrial function. CONCLUSIONSOTUD5 may ameliorate MASH progression via VDAC2-maintained mitochondrial homeostasis. Targeting OTUD5 may be a viable MASH-treatment strategy.