Dietary sugars, fructose and glucose, promote hepatic de novo lipogenesis and modify the effects of a high-fat diet (HFD) on the development of insulin resistance. Here, we show that fructose and glucose supplementation of an HFD exert divergent effects on hepatic mitochondrial function and fatty acid oxidation. This is mediated via three different nodes of regulation, including differential effects on malonyl-CoA levels, effects on mitochondrial size/protein abundance, and acetylation of mitochondrial proteins. HFD- and HFD plus fructose-fed mice have decreased CTP1a activity, the rate-limiting enzyme of fatty acid oxidation, whereas knockdown of fructose metabolism increases CPT1a and its acylcarnitine products. Furthermore, fructose-supplemented HFD leads to increased acetylation of ACADL and CPT1a, which is associated with decreased fat metabolism. 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.