Vitamin D is predominantly sequestered in adipose tissue, where it is slowly mobilized under conditions of deficiency in vivo. However, the kinetics of its uptake, release, and interaction with its major metabolites, 25(OH)D and 1,25(OH)D, remain poorly understood. Given the close relationship between obesity, low-grade chronic inflammation, and disrupted vitamin D metabolism, a clearer understanding of these dynamics in adipocytes is essential. Thus, we sought to characterize time-dependent uptake and metabolites in differentiated human adipocytes. Human pre-adipocytes were differentiated in vitro and exposed to either vitamin D and 1,25(OH)D or the combination of vitamin D, 25(OH)D and 1,25(OH)D. Intracellular concentrations were quantified through HPLC at various time points. A separate efflux experiment assessed vitamin D release under basal and isoproterenol-stimulated conditions using H-vitamin D and scintillation counting. Vitamin D uptake showed a gradual and sustained increase over 96 h, suggesting ongoing accumulation within lipid-rich compartments. In contrast, 25(OH)D and 1,25(OH)D peaked rapidly within the first hour and declined sharply. Isoproterenol stimulation significantly enhanced vitamin D release into the extracellular medium from the adipocytes, indicating increased efflux during lipolytic activation. Adipocytes selectively retain vitamin D while rapidly clearing its hydroxylated forms. These findings highlight the distinct intracellular handling of vitamin D metabolites and suggest that tailored supplementation strategies-particularly in individuals with excess adiposity-may improve bioavailability and metabolic efficacy.