Vitamin D influences skeletal health as well as other aspects of human health. Even when the most obvious sources of variation such as solar UVB exposure, latitude, season, clothing habits, skin pigmentation and ethnicity are selected for, variation in the serum 25-hydroxy vitamin D (25(OH)D) response to UVB remains extensive and unexplained. Our study assessed the inter-personal variation in 25(OH)D response to UVR and the maximal obtainable 25(OH)D level in 22 healthy participants (220 samples) with similar skin pigmentation during winter with negligible ambient UVB. Participants received identical UVB doses on identical body areas until a maximal level of 25(OH)D was reached. Major inter-personal variation in both the maximal obtainable UVB-induced 25(OH)D level (range 85–216 nmol l−1, mean 134 nmol l−1) and the total increase in 25(OH)D (range 3–139 nmol l−1, mean 48 nmol l−1) was found. A linear model including measured 25(OH)D baselines as personal intercepts explained 54.9% of the variation. By further including personal slopes in the model, as much as 90.8% of the variation could be explained. The explained variation constituted by personal differences in slopes thus represented 35.9%. Age, vitamin D receptor gene polymorphisms, height and constitutive skin pigmentation (a skin area not exposed to UVB) explained 15.1% of this variation. Despite elimination of most known external sources of variation, our study demonstrated inter-personal variation corresponding to an observed maximal difference of 136 nmol l−1 in the total increase of 25(OH)D and 131 nmol l−1 in the maximal level of 25(OH)D.