Fundamental knowledge gaps in relation to the 3 epimer of 25-hydroxycholecalciferol [3-epi-25(OH)D₃] limit our understanding of its relevance for vitamin D nutrition and health. The aims of this study were to characterize the 3-epi-25(OH)D₃ concentrations in a nationally representative sample of adults and explore its determinants. We also used data from a recent randomized controlled trial (RCT) of supplemental cholecalciferol (vitamin D₃) conducted in winter in older adults to directly test the impact of changes in vitamin D status on serum 3-epi-25(OH)D3 concentrations. Serum 25-hydroxycholecalciferol [25(OH)D₃] and 3-epi-25(OH)D₃ concentrations (via LC-tandem mass spectrometry) from our vitamin D₃ RCT in adults (aged ≥50 y) and data on dietary, lifestyle, and biochemical characteristics of participants of the recent National Adult Nutrition Survey in Ireland (aged 18-84 y; n = 1122) were used in the present work. In the subsample of participants who had serum 3-epi-25(OH)D₃ concentrations greater than the limit of quantification (n = 1082; 96.4%), the mean, 10th, 50th (median), and 90th percentile concentrations were 2.50, 1.05, 2.18, and 4.30 nmol/L, respectively, whereas the maximum 3-epi-25(OH)D₃ concentration was 15.0 nmol/L. A regression model [explaining 29.9% of the variability in serum 3-epi-25(OH)D₃] showed that age >50 y, vitamin D supplement use, dietary vitamin D, meat intake, season of blood sampling, and sun exposure habits were significant positive determinants, whereas increasing waist circumference and serum 25-hydroxyergocalciferol concentration were significant negative determinants. The RCT data showed that mean serum 25(OH)D₃ and 3-epi-25(OH)D₃ concentrations increased (49.3% and 42.1%, respectively) and decreased (-28.0% and -29.1%, respectively) significantly (P < 0.0001) with vitamin D₃ (20 μg/d) and placebo supplementation, respectively, over 15 wk of winter. In conclusion, we provide data on serum 3-epi-25(OH)D₃ in a nationally representative sample of adults. Our combined observational and RCT data might suggest that both dietary supply and dermal synthesis of vitamin D₃ contribute to serum 3-epi-25(OH)D₃ concentration.