Mitochondrial fatty acid oxidation (β-oxidation) is an essential metabolic process for energy production in eukaryotic cells, but the regulatory mechanisms of this pathway are largely unknown. In the present study, we found that several enzymes involved in β-oxidation are associated with CLPX, the AAA + unfoldase that is a component of the mitochondrial matrix protease ClpXP. Deletion of the CLPX gene increased β-oxidation activity in human hepatic cells but did not change the protein levels of enzymes involved in β-oxidation. Moreover, glucagon treatment resulted in decreased CLPX levels and increased β-oxidation activity in these cells, while the expression levels of enzymes involved in β-oxidation were not altered. Coimmunoprecipitation experiments revealed that glucagon treatment induced the dissociation of several β-oxidation-related enzymes from CLPX, suggesting that CLPX regulates β-oxidation by modulating the protein complex formation of enzymes catalyzing β-oxidation.