Hyperhomocysteinemia induces oxidative stress and chronic inflammation (both of which are catabolic to bone and muscle); thus, we examined the association between homocysteine and body composition and physical function in middle-aged and older adults. Data from the National Health and Nutrition Examination Survey was used to build regression models. Plasma homocysteine (fluorescence immunoassay) was used as the exposure and bone mineral density (BMD; dual-energy X-ray absorptiometry; DXA), lean mass (DXA), knee extensor strength (isokinetic dynamometer; newtons) and gait speed (m/s) were used as outcomes. Regression models were adjusted for confounders (age, sex, race/Hispanic origin, height, fat mass %, physical activity, smoking status, alcohol intakes, cardiovascular disease, diabetes, cancer and vitamin B12). All models accounted for complex survey design by using sampling weights provided by NHANES. 1480 adults (median age: 64 years [IQR: 56, 73]; 50.3% men) were included. In multivariable models, homocysteine was inversely associated with knee extensor strength (β = 0.98, 95% CI 0.96, 0.99, p = 0.012) and gait speed (β = 0.85, 95% CI 0.78, 0.94, p = 0.003) and borderline inversely associated with femur BMD (β = 0.84, 95% CI 0.69, 1.03, p = 0.086). In the sub-group analysis of older adults (≥ 65 years), homocysteine was inversely associated with gait speed and femur BMD (p < 0.05) and the slope for knee extensor strength and whole-body BMD were in the same direction. No significant associations were observed between homocysteine and total or appendicular lean mass in the full or sub-group analysis. We found inverse associations between plasma homocysteine and muscle strength/physical function, and borderline significant inverse associations for femur BMD.