The accumulation of fatty acids in the liver associated with obesity condition is also known as nonalcoholic fatty liver disease (NAFLD). The impaired fat oxidation in obesity condition leads to increased hepatic fat accumulation and increased metabolic syndrome risk. On the other hand, physical exercise has been demonstrated as a potent strategy in the prevention of NAFLD. Also, these beneficial effects of exercise occur through different mechanisms. Recently, the Cdc2-like kinase (CLK2) protein was associated with the suppression of fatty acid oxidation and hepatic ketogenesis. Thus, obese animals demonstrated elevated levels of hepatic CLK2 and decreased fat acid oxidation. Here, we explored the effects of chronic physical exercise in the hepatic metabolism of obese mice. Swiss mice were distributed in Lean, Obese (fed with high-fat diet during 16 weeks) and Trained Obese group (fed with high-fat diet during 16 weeks and exercised (at 60% exhaustion velocity during 1 h/5 days/week) during 8 weeks. In our results, the obese animals showed insulin resistance, increased hepatic CLK2 content and increased hepatic fat accumulation compared to the Lean group. Otherwise, the chronic physical exercise improved insulin resistance state, prevented the increased CLK2 in the liver and attenuated hepatic fat accumulation. In summary, these data reveal a new protein involved in the prevention of hepatic fat accumulation after chronic physical exercise. More studies can evidence the negative role of CLK2 in the control of liver metabolism, contributing to the improvement of insulin resistance, obesity, and type 2 diabetes.