The relation between ACTN3 R577X polymorphism and muscle mass in women has been reported, but its relation to age remains unclear. We investigated the relationship between ACTN3 R577X polymorphism and muscle mass in both middle-aged and elderly women. Two age groups (middle-aged and older) were formed among 162 healthy, nontraining Japanese women (mean ± SE, 58.6 ± 0.8 year). Their midthigh-muscle cross-sectional area (CSA) was assessed using magnetic resonance imaging, revealing no difference in thigh-muscle CSA among ACTN3 R577X genotypes in the middle-aged group (XX, 87.3 ± 2.5 cm(2); RR&RX, 86.1 ± 1.7 cm(2), p=0.7). In contrast, the XX genotype in the older group had a smaller thigh-muscle CSA adjusted to body weight than the RR&RX genotypes (XX, 67.8 ± 2.0 cm(2); RR&RX, 72.5 ± 1.2 cm(2), p<0.05). The present study showed an association between ACTN3 R577X polymorphism and smaller thigh-muscle CSA in a group of elderly women but not in a group of middle-aged women.