Glucose metabolism in lower limb muscles during walking has an important role in gait efficiency and endurance. The purpose of this study was to identify differences in muscle activity during walking between healthy young and older adults using positron emission tomography (PET) and [18F]fluorodeoxyglucose (FDG). Ten healthy young men (24 ± 2 years) and 10 healthy older men (76 ± 2 years) participated in this study. An FDG PET assessment of each subject was conducted after 50 min of treadmill walking. The images of glucose metabolism in 18 regions of interest were estimated from the standardized uptake value (SUV). The older adults showed a significantly higher FDG uptake in the semitendinosus, biceps femoris, iliacus, gluteus minimus, gluteus medius, and gluteus maximus muscles than the young adults: FDG uptake ratios of SUV in the old to SUV in the young were 3.02, 3.19, 1.66, 1.64, 3.68, and 3.05, respectively. During walking, the FDG uptake in older adults was higher in hamstrings and deep layer hip muscles than that in young adults. These results suggest that intervention to facilitate efficient muscle activity during walking should be practiced to improve gait endurance in older adults with impaired walking patterns.