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 (vitaminD3) conductedinwinter inolder adults to directly test the impact of changes invitaminD status onserum 3-epi-25(OH)D3 concentrations.Serum25-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 NationalAdultNutrition Survey in Ireland (aged 18-84 y;n=1122)were usedin the present work. Inthesubsampleofparticipantswhohadserum3-epi-25(OH)D3concentrations greater thanthelimitof 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 serum3-epi-25(OH)D3] showed that age >50 y, vitaminDsupplement 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 (228.0% and229.1%, respectively) significantly (P<0.0001) with vitaminD3 (20mg/d) andplacebo supplementation, respectively, over 15wkofwinter. 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.