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Simple Summary: Vitamin D biology in equids is unique and poorly understood. Naturally managed (grazing) horses rely on dietary vitamin D2 (ergocalciferol) to provide adequate vitamin D, because endogenous ultraviolet radiation-mediated synthesis of vitamin D3 (cholecalciferol) is ineffective in this species. To test the hypothesis that the management of stabled, non-grazing racehorses is a risk factor for low vitamin D status, the vitamin D status of non-grazing Thoroughbred racehorses in Hong Kong (HK) and grazing Thoroughbred racehorses in the United Kingdom (UK) was compared. The HK horses had lower serum concentrations of 25-hydroxyvitamin D2 (25OHD2) and total 25-hydroxyvitamin D (total 25OHD: the index of vitamin D status), reflecting reduced dietary vitamin D2 intake. These data indicate that HK racehorses required dietary vitamin D3 supplementation to maintain adequate vitamin D status. The inverse relationship between the serum concentrations of 25OHD2 and 25OHD3, previously identified in humans, was observed for the first time in horses, indicating that further study is needed to determine the optimal form of dietary vitamin D supplementation for Thoroughbred racehorses. 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. [ABSTRACT FROM AUTHOR]