The activity of respiratory chain enzymes in a rat's masseter muscle changes as the animal ages; however, there is little information about the RNA transcript levels of mitochondrial enzymes in klotho mutant mice as they age. We measured the activities of NADH-ferricyanide oxidoreductase and NADH-O2 oxidoreductase, and the RNA transcript levels of NADH dehydrogenase, the mitochondrial isoform of ND1, the nuclear isoforms of the 51 kDa and 75 kDa subunits of Complex I, the nuclear isoform of cytochrome c, and the mitochondrial isoform of beta subunits of ATPase (Complex V). In addition, we measured the RNA transcript levels of catalase (CAT) and superoxide dismutase (SOD), which are associated with antioxidant proteins. Moreover, we measured ATP concentrations using a luciferin-luciferase assay, and we determined the amount of cytochrome c associated with mitochondria in both klotho mutant mice and wild-type mice. However, the mRNA levels of cytochrome c and Complex V components, the mRNA levels of CAT, SOD, and apoptosis-inducing factor (Aifm), and the protein level of cytochrome c remained constant as klotho mutant mice aged from 5 weeks to 7 weeks. In wild-type mice, these components (except for those of Complex I) increased over time. NADH-ferricyanide oxidoreductase and NADH-O2 oxidoreductase activities decreased in klotho mutant mice as they aged from 5 weeks to 7 weeks. A few large mitochondria were scattered between myofibrils, and 7-week-old klotho mutant mice displayed an increased number of irregular mitochondria with fewer cristae. Our results indicate that the klotho protein plays a role in the diminished functional adaptability of enzymes in the masseter muscle of klotho mutant mice throughout the aging process.