Background: The optimal amount of vitamin D required for proper functioning of the immune system differs from the amount necessary for bone homeostasis. Furthermore, vitamin D metabolism varies among horses. Nevertheless, there is a dearth of information regarding reference values for vitamin D in horses, particularly in the Turkmen breed. The primary objectives of this study were to determine the concentrations of 25-hydroxyvitamin D (25(OH)Vit D) in Turkmen, Thoroughbred, and mixed Turkmen×Thoroughbred horses and to explore the effects of various factors on it and its relationship with serum biochemical variables and signalment data. Methods: For the measurement of 25(OH)Vit D, 90 healthy horses (min age: 6 months, max age: 10 years) that were stable at north of Iran (37°17’ N, 55°18’ E) were selected for clinical examination and complete blood biochemistry analysis. The horses were categorized into different groups based on sex, season (spring, n=45; autumn, n=45), breed (Turkmen, Thoroughbred, and mixed Turkmen×Thoroughbred), age (less than 2 years, 2-8 years, older than 8), and skin color. Sampling was performed only once from each horse. Results: In this study, the median serum concentration of 25(OH)Vit D in the sampled horses was 17.42 ng/ml (IQR 9.82-30.85). The serum 25(OH)Vit D concentration was significantly lower in autumn (median 15.83 ng/ml and IQR 8.67-54.68) than in spring (median 18.02 ng/ml and IQR 13.77-27.54) and was also lower in Turkmen horses (median 15.83 ng/ml and IQR 11.63-23.12) than in mixed breed horses (median 17.72 ng/ml and IQR 8.94-51.67) (p≤0.05). Conclusion: According to the results of the present study, season had significant effect on the serum 25(OH)Vit D concentration and this difference was also observed between Turkmen horses and mixed bred horses (p≤0.05). It is unclear that the time of sun exposure or vitamin D content of diet cause seasonal difference of 25(OH)Vit D concentration.

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