It is known that a high-fat diet induces an increase in mitochondrial biogenesis in skeletal muscle. To examine the time course of decrease in mitochondrial biogenesis in skeletal muscle after discontinuing a high-fat diet feeding, C57BL/6 mice were fed a high-fat diet for 4 wk and then switched to the control diet for another 3 or 7 d. During the high-fat diet withdrawal period, the protein content of the mitochondrial respiratory chain decreased faster than the fatty acid oxidation enzymes. The mitochondrial DNA copy number remained high for at least 1 wk after withdrawing the high-fat diet. These results suggested that after switching to the control diet following a period of high-fat diet, the increased mitochondrial biogenesis levels are maintained for a few days, and the rate of decline is divergent between the different mitochondrial components.