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There is a paucity of data relating to the vitamin D status of racehorses. We hypothesised that the management of racehorses in Hong Kong (HK) predisposes to low vitamin D status unless they receive dietary supplementation. Serum concentrations of 25-hydroxyvitamin D₂ (25OHD₂), 25-hydroxyvitamin D₃ (25OHD₃) and total 25-hydroxyvitamin D (total 25OHD) for 79 non-grazing HK racehorses were compared with those for 22 racehorses training in the United Kingdom (UK) that grazed for ≥1 h/d, and for which published data exists. A nested group of 41 HK horses was sampled twice to determine the effect of the duration in HK on vitamin D status. The HK horses had significantly lower serum concentrations of total 25OHD and 25OHD₂ than the UK horses; 25OHD₂ was undetectable in 15/79 HK sera and serum concentrations of 25OHD₂ declined with the duration in HK. The main determinants of vitamin D status were assessed using linear regression; the retained variables were the 25OHD₃ concentration and the duration in HK. The inverse relationship between the serum concentrations of 25OHD₂ and 25OHD₃, previously identified in humans, was observed for the first time in horses. In conclusion, HK racehorses have low serum 25OHD₂ and total 25OHD concentrations and rely on D₃ supplementation to maintain adequate vitamin D status. Further study is required to determine the optimal form of dietary vitamin D supplementation for Thoroughbred racehorses.