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Elevated plasma homocysteine has been linked to reduced mobility and muscle functioning in the elderly. The relation of methylenetetrahydrofolate reductase (MTHFR) 677C → T polymorphism with these associations has not yet been studied. This study aimed to investigate (1) the association of plasma homocysteine and the MTHFR 677C → T polymorphism with muscle mass, handgrip strength, physical performance and postural sway; (2) the interaction between plasma homocysteine and the MTHFR 677C → T polymorphism.Baseline data from the B-PROOF study (n = 2919, mean age = 74.1 ± 6.5) were used. Muscle mass was measured using dual X-ray absorptiometry, handgrip strength with a handheld dynamometer, and physical performance with walking-, chair stand- and balance tests. Postural sway was assessed on a force platform. The data were analyzed using regression analyses with plasma homocysteine levels in quartiles.There was a significant inverse association between plasma homocysteine and handgrip strength (quartile 4: regression coefficient B = −1.14, 95% confidence interval (CI) = −1.96; −0.32) and physical performance score (quartile 3: B = −0.53, 95% Cl = −0.95; −0.10 and quartile 4: −0.94; 95% Cl = −1.40; −0.48) in women only, independent of serum vitamin B12 and folic acid. No association was observed between the MTHFR 677C → T polymorphism and the outcomes. High plasma homocysteine in the 677CC and 677CT genotypes, but not in the 677TT genotype, was associated with lower physical performance.Elevated plasma homocysteine concentrations are associated with reduced physical performance and muscle strength in older women. There is an urgent need for randomized controlled trials to examine whether lowering homocysteine levels might delay physical decline.