ACOT8 is a peroxisomal acyl-CoA thioesterase that catalyzes hydrolysis of acyl-CoA esters to free fatty acids and coenzyme A 1. ACOT8 exhibits broad substrate specificity, preferentially hydrolyzing longer dicarboxylic acid-CoA esters (glutaryl-, adipyl-, suberyl-, sebacyl-, and dodecanedioyl-CoA) with high activity 2. The enzyme plays critical roles in fatty acid and bile acid metabolism, supporting peroxisomal β-oxidation and energy production 1. Under energy stress conditions, hepatic ACOT8 is dramatically upregulated to convert fatty acid-derived acetyl-CoA to acetate, providing fuel for the brain and recycling CoA for sustained fatty acid oxidation and ketogenesis 3. Beyond metabolic functions, ACOT8 interacts with HIV-1 Nef protein through specific structural regions (Arg45-Phe55 and Arg86-Pro93), with Lys91 being critical for this association 4. This interaction may preserve Nef from degradation and modulate lipid composition in membrane rafts 5. Clinically, ACOT8 overexpression promotes cancer progression. Elevated ACOT8 is frequently observed in hepatocellular carcinoma, where it drives cell growth through free fatty acid metabolism 6. In pancreatic ductal adenocarcinoma, ACOT8 confers gemcitabine resistance via altered lipid metabolism and suppression of ferroptosis 7. ACOT8 is also a component of lipogenic biomarker panels for colorectal cancer diagnosis and prognosis 8.