Large parts of the population are insufficiently supplied with vitamin D, in particular when endogenous synthesis is absent. Therefore many health care providers recommend the use of vitamin D supplements. The current study aimed to investigate the efficacy of an once-daily oral dose of 20 μg vitamin D3 to improve the vitamin D status and to evaluate predictors of response.The study was conducted as a double-blind, randomized, placebo-controlled parallel trial from January till April 2013. In total, 105 subjects (20-71 years) were allocated to receive either a vitamin D3 supplement (20 μg/d) or a placebo for 12 weeks. Circulating levels of vitamin D3 metabolites such as the 25(OH)D3 and the 24,25(OH)2D3, and biomarkers of calcium and phosphate metabolism were quantified.The 25(OH)D3 serum concentrations in the placebo group decreased from 38 ± 15 nmol/L at baseline to 32 ± 14 nmol/L and 32 ± 13 nmol/L at weeks 8 and 12 of the study, respectively (p < 0.01). In the vitamin D3 group, the serum 25(OH)D3 concentration increased from 38 ± 14 nmol/L at baseline to 70 ± 15 nmol/L and 73 ± 16 nmol/L at weeks 8 and 12 of vitamin D3 supplementation (p < 0.001), respectively. As a result, 94% of the vitamin D3-supplemented participants reached 25(OH)D3 concentrations of ≥50 nmol/L and thereof 46% attained 25(OH)D3 levels of ≥75 nmol/L until the end of the study. The extent of the 25(OH)D3 increase upon vitamin D3 supplementation depended on 25(OH)D3 baseline levels, age, body weight and circulating levels of triglycerides. In contrast to 25(OH)D3, the response of 24,25(OH)2D3 to the vitamin D3 treatment was affected only by baseline levels of 24,25(OH)2D3 and age.The average improvement of 25(OH)D3 levels in individuals who received 20 μg vitamin D3 per day during the winter months was 41 nmol/L compared to individuals without supplementation. As a result almost all participants with the vitamin D3 supplementation attained 25(OH)D3 concentrations of 50 nmol/L and higher. The suitability of 24,25(OH)2D3 as a marker of vitamin D status needs further investigation. Clinical trial registration number at clinicaltrials.gov: NCT01711905.