Evidence supports the role of vitamin D in various conditions of development and ageing. Serum 25-hydroxyvitamin D (25(OH)D) is the best indicator for current vitamin D status. However, the cost of its measurement can be prohibitive in epidemiological research. We developed and validated multivariable regression models that quantified the relationships between vitamin D determinants, measured through an in-person interview, and serum 25(OH)D concentrations. A total of 200 controls participating in a population-based case–control study in Montreal, Canada, provided a blood specimen and completed an in-person interview on socio-demographic, reproductive, medical and lifestyle characteristics and personal attributes. Serum 25(OH)D concentrations were quantified by liquid chromatography–tandem MS. Multivariable least squares regression was used to build models that predict 25(OH)D concentrations from interview responses. We assessed high-order effects, performed sensitivity analysis using the lasso method and conducted cross-validation of the prediction models. Prediction models were built for users and non-users of vitamin D supplements separately. Among users, alcohol intake, outdoor time, sun protection, dose of supplement use, menopausal status and recent vacation were predictive of 25(OH)D concentrations. Among non-users, BMI, sun sensitivity, season and recent vacation were predictive of 25(OH)D concentrations. In cross-validation, 46–47 % of the variation in 25(OH)D concentrations were explained by these predictors. In the absence of 25(OH)D measures, our study supports that predicted 25(OH)D scores may be used to assign exposure in epidemiological studies that examine vitamin D exposure.