Background: Vitamin D supplementation is proposed as a potential treatment strategy to counteract functional decline in older adults. However, data from randomized trials are either limited or inconsistent. Objective: This study investigated the effect of daily supplementationwith 25-hydroxycholecalciferol [25(OH)D3] or cholecalciferol (vitamin D3) on muscle strength and physical performance in older adults. Methods: This was a randomized, double-blind, placebo-controlled trial of 6 mo including 78 prefrail or frail (according to the Fried criteria), community-dwelling older adults (n = 43 men) aged ≥65 y, with a baseline 25-hydroxyvitamin D [25(OH)D] concentration between 20 and 50 nmol/L. Participants were supplemented daily with 10 μg 25(OH)D3, 20 μg vitamin D3, or a placebo capsule. Serum 25(OH)D was measured by liquid chromatography-tandem mass spectrometry. The primary outcome wasmaximal isometric knee-extension strength (Biodex System 4); secondary outcomes included knee-flexion and hand grip strength, Short-Physical Performance Battery score, Timed Up and Go score, postural sway, muscle mass (dual-energy X-ray absorptiometry), and muscle fiber type and size. Results: The mean baseline serum 25(OH)D concentration was 37.7 nmol/L (95% CI: 35.4, 39.9 nmol/L). After 6 mo of supplementation, concentrations increased to 98.7 nmol/L (95% CI: 93.1, 104.4 nmol/L) in the 25(OH)D3 group and to 72.0 nmol/L (95% CI: 66.1, 77.8 nmol/L) in the vitamin D3 group, compared with 47.5 nmol/L (95% CI: 41.8, 53.3 nmol/L) in the placebo group (P-interaction < 0.01). Knee-extension strength did not significantly change in the 25(OH)D3 group (5.9 Nm; 95% CI: -6.2, 18.0 Nm), in the vitamin D3 group (5.5 Nm; 95% CI: -6.8, 17.8 Nm), or in the placebo group (1.8 Nm; 95% CI: -10.7, 14.4 Nm) (P-interaction = 0.74). Furthermore, mean changes in physical performance tests, muscle mass, and muscle fiber type and size did not differ between the groups. Conclusion: Increasing the serum 25(OH)D concentration over a period of 6 mo did not significantly change muscle strength and physical performance in prefrail and frail older adults.