The R577X polymorphism in the α-actinin-3 encoding gene (ACTN3) has been associated with elite athletic performance, and recently with differences in isometric and dynamic muscle strength and power in the general population. In this study we sought to determine the association of ACTN3 R577X genotype with muscle strength and mass phenotypes in men and women across the adult age span. Eight hundred forty-eight (n = 848) adult volunteers (454 men and 394 women) aged 22–90 yr were genotyped for ACTN3 R577X. Knee extensor (KE) shortening and lengthening peak torque values were determined using isokinetic dynamometry and fat-free mass (FFM) by dual-energy X-ray absorptiometry. Women deficient in α-actinin-3 (X/X; n = 53) displayed lower KE shortening peak torque (30°/s: 89.5 ± 3.5 vs. 99.3 ± 1.4 N·m, P = 0.011; 180°/s: 60.3 ± 2.6 vs. 67.0 ± 1.0 N·m, P = 0.019) and KE lengthening peak torque (30°/s: 122.8 ± 5.7 vs. 137.0 ± 2.2 N·m, P = 0.022; 180°/s: 121.8 ± 5.8 vs. 138.5 ± 2.2 N·m, P = 0.008) compared with R/X + R/R women (n = 341). Women X/X homozygotes also displayed lower levels of both total body FFM (38.9 ± 0.5 vs. 40.1 ± 0.2 kg, P = 0.040) and lower limb FFM (11.9 ± 0.2 vs. 12.5 ± 0.1 kg, P = 0.044) compared with R/X + R/R women. No genotype-related differences were observed in men. In conclusion, our results indicate that the absence of α-actinin-3 protein (i.e., ACTN3 X/X genotype) influences KE peak torque and FFM in women but not men.