A physiologically based pharmacokinetic (PBPK) model of vitamin D and metabolites 25(OH)D , 1,25(OH) D , and 24,25(OH) D is presented. In this study, patients with 25(OH)D plasma concentrations below 30 ng/ml were studied after a single dose of 5000 I.U. (125 g) cholecalciferol, provided with 5000 I.U. daily cholecalciferol supplementation until vitamin D replete 25(OH)D plasma concentrations above 30 ng/ml, and had serial plasma samples were collected at each phase for 14 days. Total concentrations of vitamin D and metabolites were measured by ultra-high performance liquid chromatography tandem mass spectrometry. A nine-compartment PBPK model was built using MATLAB to represent the triphasic study nature (insufficient, replenishing, and sufficient). The stimulatory and inhibitory effect of 1,25(OH) D were incorporated by fold-changes in the primary metabolic enzymes CYP27B1 and CYP24A1, respectively. Incorporation of dynamic adipose partition coefficients for vitamin D and 25(OH)D and variable enzymatic reactions aided in model fitting. Measures of model predictions agreed well with data from metabolites, with 97%, 88%, and 98% of the data for 25(OH)D , 24,25(OH) D , and 1,25(OH) D , respectively, within twofold of unity ( values between 0.5 and 2.0). Bootstrapping was performed and optimized parameters were reported with 95% confidence intervals. This PBPK model could be a useful tool for understanding the connections between vitamin D and its metabolites under a variety of clinical situations. SIGNIFICANCE STATEMENT: This study developed a physiologically based pharmacokinetic (PBPK) model of vitamin D and metabolites for patients moving from an insufficient to a repleted state over a period of 16 weeks.