Abstract Context: Vitamin D deficiency is common among older people and can cause mineralization defects, bone loss, and muscle weakness. Objective: The aim of this study was to investigate the association of serum 25-hydroxyvitamin D (25-OHD) concentration with current physical performance and its decline over 3 yr among elderly. Design: The study consisted of a cross-sectional and longitudinal design (3-yr follow-up) within the Longitudinal Aging Study Amsterdam. Setting: An age- and sex-stratified random sample of the Dutch older population was used. Other Participants: Subjects included 1234 men and women (aged 65 yr and older) for cross-sectional analysis and 979 (79%) persons for longitudinal analysis. Main Outcome Measure(s): Physical performance (sum score of the walking test, chair stands, and tandem stand) and decline in physical performance were measured. Results: Serum 25-OHD was associated with physical performance after adjustment for age, gender, chronic diseases, degree of urbanization, body mass index, and alcohol consumption. Compared with individuals with serum 25-OHD levels above 30 ng/ml, physical performance was poorer in participants with serum 25-OHD less than 10 ng/ml [regression coefficient (B) = −1.69; 95% confidence interval (CI) = −2.28; −1.10], and with serum 25-OHD of 10–20 ng/ml (B = −0.46; 95% CI = −0.90; −0.03). After adjustment for confounding variables, participants with 25-OHD less than 10 ng/ml and 25-OHD between 10 and 20 ng/ml had significantly higher odds ratios (OR) for 3-yr decline in physical performance (OR = 2.21; 95% CI = 1.00–4.87; and OR = 2.01; 95% CI = 1.06–3.81), compared with participants with 25-OHD of at least 30 ng/ml. The results were consistent for each individual performance test. Conclusions: Serum 25-OHD concentrations below 20 ng/ml are associated with poorer physical performance and a greater decline in physical performance in older men and women. Because almost 50% of the population had serum 25-OHD below 20 ng/ml, public health strategies should be aimed at this group.