PURPOSESupplementation with dietary forms of vitamin D is commonplace in clinical medicine, elite athletic cohorts, and the general population, yet the response of all major vitamin D metabolites to high doses of vitamin D is poorly characterized. We aimed to identify the responses of all major vitamin D metabolites to moderate- and high-dose supplemental vitamin D3. METHODSA repeated-measures design was implemented in which 46 elite professional European athletes were block randomized based on their basal 25OHD concentration into two treatment groups. Athletes received either 35,000 or 70,000 IU·wk vitamin D3 for 12 wk, and 42 athletes completed the trial. Blood samples were collected for 18 wk to monitor the response to supplementation and withdrawal from supplementation. RESULTSBoth doses led to significant increases in serum 25OHD, and 1,25OH2D3. 70,000 IU·wk also resulted in a significant increase of the metabolite 24,25OH2D at weeks 6 and 12 that persisted after supplementation withdrawal at week 18, despite a marked decrease in 1,25OH2D3. Intact parathyroid hormone was decreased in both groups by week 6 and remained suppressed throughout the trial. CONCLUSIONSHigh-dose vitamin D3 supplementation (70,000 IU·wk) may be detrimental for its intended purposes because of increased 24,25OH2D production. Rapid withdrawal from high-dose supplementation may inhibit the bioactivity of 1,25OH2D3 as a consequence of sustained increases in 24,25OH2D that persist as 25OHD and 1,25OH2D concentrations decrease. These data imply that lower doses of vitamin D3 ingested frequently may be most appropriate and gradual withdrawal from supplementation as opposed to rapid withdrawal may be favorable.