Context:Studies examining whether vitamin D supplementation increases muscle mass or muscle-specific vitamin D receptor (VDR) concentration are lacking.Objective:To determine whether vitamin D3 4000 IU/d alters muscle fiber cross-sectional area (FCSA) and intramyonuclear VDR concentration over 4 months.Design and Setting:Randomized, double-blind, placebo-controlled study in a single center.Participants:21 mobility-limited women (aged ≥65 years) with serum 25-hydroxyvitamin D (25OHD) levels 22.5-60 nmol/L.Main Outcome Measures:Baseline and 4-month FCSA and intramyonuclear VDR were measured from vastus lateralis muscle cross-sections probed for muscle fiber type (I/IIa/IIx) and VDR using immunofluorescence.Results:At baseline, mean (±SD) age was 78±5 years; body mass index (BMI) was 27±5 kg/m(2); 25OHD was 46.3±9.5 nmol/L; and a short physical performance battery score was 7.95±1.57 out of 12. At 4 months, 25OHD level was 52.5±17.1 (placebo) vs. 80.0±11.5 nmol/L (VD; P<0.01) and change in 25OHD level was strongly associated with percent change in intramyonuclear VDR concentration independent of group (r=0.87, P<0.001). By treatment group, percent change in intramyonuclear VDR concentration was 7.8±18.2% (placebo) vs. 29.7±11.7% (VD; P=0.03) with a more pronounced group difference in type II vs. I fibers. Percent change in total (type I/II) FCSA was -7.4±18.9% (placebo) vs. 10.6±20.0% (VD; P=0.048).Conclusion:Vitamin D3 supplementation increased intramyonuclear VDR concentration by 30% and increased muscle fiber size by 10% in older, mobility-limited, vitamin D-insufficient women. Further work is needed to determine whether the observed effect of vitamin D on fiber size is mediated by the VDR and to identify which signaling pathways are involved.