In addition to the known effects of vitamin D on the skeletal system, its effects on other systems have been revealed by studies, and clinical and animal studies have been carried out frequently in recent years. Although there are various forms of vitamin D, the most active and effective is calstriol, known as 1,25(OH)2D. The aim of this study is to determine the effective and safe dose for the evaluation of the effect of calstriol on pain threshold in rats. In the experiment, 32 Wistar Albi-no male rats, 2.5 months old (240-260 gr) were used. The animals were randomly divided into 4 groups with 8 animals in each group. Groups; control, vitamin D= 1 µg/kg, vitamin D= 10 µg/kg and vitamin D= 100 µg/kg. Tail flick and hot plate tests were used to evaluate the pain threshold. Measurements were taken at the 0th minute before the drug administration and at the 30th, 60th and 90th minutes after the drug administration, and the times were recorded in seconds. Vitamin D= 10 µg/kg administered group significantly prolonged the pain tolerance time in both tail flick and hot plate test compared to the other groups (p˂0.05). Thanks to the effective dose obtained as a result of our study, experimental pain models using calstriol will be facilitated and further studies will be contributed without wasting extra time and animal loss for the researchers.