Background: Vitamin D deficiency is associated with active Crohn’s disease (CD). However, it remains unclear if lower 25-hydroxyvitamin D [25(OH)D] concentration is the cause, or consequence, of intestinal inflammation. Existing literature has focused on circulating 25(OH)D rather than the active metabolite 1,25(OH) 2 D, or its breakdown product, 24,25(OH) 2 D. We aimed to characterise vitamin D metabolism in a cohort of patients with active and inactive CD. Methods: Fifty-four patients with CD and not on corticosteroids or vitamin D supplements, were enrolled in a 6-month prospective cohort study. Sera were collected on enrolment and at 6 months and tested for 25(OH)D, 1,25(OH) 2 D, 24,25(OH) 2 D using liquid chromatography tandem mass spectroscopy as well as vitamin-D-binding protein. Results: There were no differences in 25(OH)D or 1,25(OH) 2 D levels between participants with active versus inactive disease. Levels of 24,25(OH) 2 D were significantly lower in those with active compared with inactive disease (mean 3.9 versus 6.0 µmol/l; p = 0.007) and therefore the ratio of 25(OH)D:24,25(OH) 2 D was higher (mean 17.3 versus 11.1; p = 0.001). In those patients with active disease who achieved remission, there was a mean increase in 25(OH)D of 32.3 nmol/l (i.e. to a level in the sufficient range) and 24,25(OH) 2 D of 2.1 µmol/l. These increases were not seen in patients with persistently active or inactive disease. Conclusion: Levels of 24,25(OH) 2 D, but not 25(OH)D, were lower in patients with active CD, and spontaneously increased with resolution of underlying inflammation. The utility of 24,25(OH) 2 D as a biomarker of disease activity and vitamin D status in CD warrants further exploration.