Objective: To investigate the effect of interleukin-25 (IL-25) on ovalbumin (OVA) induced atopic dermatitis of mice, and the significance of regulating IL-25. Methods: In this study, 90 healthy male 6-week-old specific pathogen free (SPF) BALB/c mice were divided into 6 groups (15 in each group): ① subcutaneous injection of phosphate buffered saline (PBS) group (normal control group); ② subcutaneous injection of mouse IL-25 group (IL-25 group); ③ subcutaneous injection of anti-mouse IL-25 monoclonal antibody (anti-IL-25 group), each group received subcutaneous injection once a day for 1 week, 2 weeks apart, repeated daily subcutaneous injections for 1 week, 2 weeks apart, and repeated daily subcutaneous injections for 1 week, for a total of 7 weeks; ④ OVA treated group (model group); ⑤ OVA treated and IL-25 subcutaneous injection group (IL-25 treated dermatitis group); ⑥ OVA treated and anti-mouse IL-25 monoclonal antibody injection group (anti-IL-25 treated dermatitis group). The ⑤ and ⑥ groups in the process of treatment with OVA, IL-25 or anti-IL-25 antibody were given in the same way as the ② and ③ groups. Scratching behavior and skin performance of the mice were recorded during the seven-week-treatment. Twenty four hours after the final treatment, blood was taken from the mouse heart, and the serum was separated to detect the total IgE, IL-4, IL-5, IL-13, etc. The skin samples of the treatment sites were used for hematoxylin-eosin (HE) staining, immunohistochemistry, real-time PCR and Western blot detections. A single factor (ANOVA) analysis of variance was used to compare the differences in various indicators between the groups. Results: The frequency of scratches in the IL-25 treated dermatitis group was higher than that in the model group, and the scratching behavior of the anti-IL-25 treated dermatitis group was significantly lower than that in the model group. The appearance of atopic dermatitis, thickening of the epidermis and the degree of dermal inflammation in the IL-25 treated dermatitis group were more serious than those in the model group and the anti-IL-25 treated dermatitis group. The levels of serum IgE, IL-4, IL-5, and IL-13 in the IL-25 treated dermatitis group were significantly higher than that in the model group and the anti-IL-25 treated dermatitis group. There were significantly more CD4⁺ T cells in the dermis of IL-25 treated dermatitis group than that in the anti-IL-25 treated dermatitis group. The expression levels of filaggrin and defensin β2 proteins in the IL-25 treated dermatitis group were significantly lower than those in the model group and the anti-IL-25 treated dermatitis group. Conclusion: In the OVA induced atopic dermatitis mice model, IL-25 can significantly promote the damage of the epidermal barrier function and aggravate the OVA-induced dermatitis. Antagonizing IL-25 can alleviate OVA induced dermatitis to a certain extent.