Objective: The current study aimed to examine homocysteine in relation to different aspects of physical functioning. Design, setting and participants: Cross-sectional and longitudinal data (3-years follow-up) from the Longitudinal Aging Study Amsterdam (LASA) were used. The study was performed in persons aged >/= 65 years (N= 1301 after imputation). Measurements: Different measures of physical functioning, including muscle mass, grip strength, functional limitations, and falling were regarded as outcomes. Gender and serum creatinine level were investigated as effect modifiers. Results: Results were stratified by gender. In men, higher homocysteine levels were associated with lower grip strength (Quartile 4: regression coefficient (B)= -3.07 (-4.91; -1.22)), and more functional limitations at baseline (Quartile 4: B= 1.15 (0.16-2.14)). In women, higher homocysteine levels were associated with more functional limitations after 3 years (Quartile 4: B= 1.19 (0.25; 2.13)). Higher homocysteine levels were not associated with low muscle mass or falling. Conclusions: These data suggest an inverse association of homocysteine levels with functional limitations in older men and women, and with muscle strength in older men.