Context: Vitamin D is known to influence muscle health. A reduction in muscle mass increases the risk of functional limitations among older individuals. Objective: The aim of this study was to examine the relationship between vitamin D status and functional limitations. Design, Setting, and Participants: Two independent cohorts of the Longitudinal Aging Study Amsterdam were used. Participants were aged 65 to 88 years (older cohort, n = 1237; baseline 1995) and 55 to 65 years (younger cohort, n = 725; baseline 2002). Main Outcome Measures: Questions on the ability and degree of difficulty to perform 6 functions of daily life were asked. Results: Of the participants, 56% in the older cohort and 30% in the younger cohort had ≥1 limitation. Vitamin D deficiency (25-hydroxyvitamin D level of <20 ng/mL) compared with the value in the reference group (>30 ng/mL) was related to the presence of functional limitations at baseline (odds ratio [OR] = 1.7; 95% confidence interval [CI], 1.2–2.5 and OR = 2.2; 95% CI 1.3–3.7 for the older and younger cohorts, respectively). In the older cohort, vitamin D deficiency was associated with an increase in limitations at 3 years (OR = 2.0; 95% CI, 1.1–3.5), whereas vitamin D deficiency in the younger cohort was associated with an increase in limitations at 6 years (OR = 3.3; 95% CI, 1.1–10.1). Analyses were adjusted for confounders. Conclusion: Vitamin D status is associated with functional limitations cross-sectionally and longitudinally in individuals aged 55 to 65 years and those 65 years and older. The possible association of vitamin D with functional limitations is present after a shorter follow-up time in the oldest age group compared with the younger age group.