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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.