• Addition of fructose to a high-fat diet increases hepatic malonyl-CoA more than glucose
•Knockdown of the fructose metabolizing gene ketohexokinase increases CTP1a levels
•Fructose supplementation alters mitochondrial size and function
•Dietary fructose induces acetylation of ACADL and CPT1a to modify fat oxidation
In summary, dietary fructose, but not glucose, supplementation of HFD impairs mitochondrial size, function, and protein acetylation, resulting in decreased fatty acid oxidation and development of metabolic dysregulation.