Objective: To investigate the role of leukotriene B4 (LTB4) and 25-hydroxyvitamin D3 in recurrent respiratory infections in preschool children. Methods: One hundred and thirty-eight preschool children with recurrent respiratory tract infection who visited the undergraduate room for 2 consecutive years were selected as the observation group. Meanwhile, 84 healthy children who had physical examination at the same time were selected as the control group. There was no significant difference in gender ratio between the control group and the observation group (P>0.05). Peripheral blood serum levels of LTB4 and 25-hydroxyvitamin D3 were measured by ELISA and the correlation was analyzed. Human airway epithelial cell line H292 was studied, and an inflammatory cell model was constructed using LPS to analyze the effect of 25-hydroxyvitamin D3 level on LTB4 level. Results: Peripheral blood serum 25-hydroxyvitamin D3 level of children with recurrent respiratory tract infection was significantly lower than that of the control group (P<0.001), while peripheral blood serum LTB4 level was significantly higher than that of healthy control group (P<0.001). There was a significant negative correlation between the average number of upper respiratory tract infection and 25-hydroxyvitamin D3 in peripheral blood of children with upper respiratory tract infection (r=−0.502, P=0.019). The mean number of upper respiratory tract infection and the peripheral blood serum LTB4 level of children with upper respiratory tract infection showed a significant positive correlation (r=0.852, P<0.001). The serum levels of LTB4 and 25-hydroxyvitamin D3 showed a significant negative correlation (r=−0.431, P=0.024). Vitro cell experiments showed that 25-hydroxyl vitamin D3 levels inhibited LTB4 levels. Conclusion: The deficiency of serum 25-hydroxyvitamin D3 in the recurrent upper respiratory tract infection of preschool children can lead to increased LTB4.