Context: Vitamin D deficiency and frailty are common with aging, but the association between these conditions is uncertain. Objective: To determine the association between 25-hydroxyvitamin D (25(OH)D) levels and prevalent and incident frailty status among older women. Design: Cross-sectional and longitudinal analyses of a prospective cohort study. Setting: Four U.S. centers. Participants: 6307 women aged ≥69 years. Main Outcome Measures: Frailty status classified as robust, intermediate stage, or frail at baseline; and robust, intermediate stage, frail, or dead (all-cause mortality) at follow-up an average of 4.5 years later. Results: At baseline, there was a U-shaped association between 25(OH)D level and odds of frailty with the lowest risk among women with levels 20.0-29.9 ng/ml (referent group). Compared with this group, the odds of frailty were higher among those with levels < 15.0 ng/ml [multivariable odds ratio (MOR) 1.47, 95% confidence interval (CI), 1.19-1.82], those with levels 15.0-19.9 ng/ml (MOR 1.24, 95% CI 0.99-1.54), and those with levels ≥30 ng/ml (MOR 1.32, 95% CI 1.06-1.63). Among 4551 nonfrail women at baseline, the odds of frailty/death (vs. robust/intermediate) at follow-up appeared higher among those with levels 15.0-19.9 ng/ml (MOR 1.21, 95% CI 0.99-1.49), but the CI overlapped 1.0. The odds of death (vs. robust/intermediate/frail at follow-up) was higher among those with levels < 15.0 ng/ml (MOR 1.40, 95% CI 1.04-1.88) and those with levels 15.0-19.9 ng/ml (MOR 1.30, 95% CI 0.97-1.75), although the latter association did not quite reach significance. Conclusion: Lower (<20 ng/ml) and higher (≥30 ng/ml) levels of 25(OH)D among older women were moderately associated with a higher odds of frailty at baseline. Among nonfrail women at baseline, lower levels (<20 ng/ml) were modestly associated with an increased risk of incident frailty or death at follow-up.