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Background Allergen-specific immunotherapy can induce immune tolerance to specific allergens by regulating immune status of individuals. However, its clinical application is limited due to individual differences in efficacy among patients and un-confirmed safety. 1,25 Dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to be involved in a variety of physiological processes, including immune response regulation. In the present study we explored the role of 1,25(OH)2D3 pretreatment for immunotherapy.Methods Seventy-five BALB/c mice were randomly divided into five groups (15 mice per group). The mouse allergic asthma model was established by intra-peritoneal injection of ovalbumin (OVA, 10 μg) and aluminium hydroxide (2 mg)as an adjuvant. Intra-peritoneal injection of 50 ng of 1,25(OH)2D3 served as a pretreatment, subcutaneous injection of OVA (100 μg) as an immunotherapy, and 1% OVA inhalation as a challenge. Histopathological analysis was performed on four mice per group. The number of cells and their classification in bronchoalvolar lavage (BAL) fluid were assayed.Levels of serum OVA-specific immunoglobulin E (slgE) and IFN-Y, IL-4, IL-5 and IL-10 in BAL fluid were measured by ELISA.Results After 1,25(OH)2D3 pretreatment, immunotherapy could significantly inhibit the infiltration of inflammatory cells into lung tissues and BAL fluid of mice with allergic asthma when compared with un-treated animals (eosinophils:(7.46±1.34)×104/ml vs. (13.41±1.67)×104/ml, P ＜0.05). In addition, levels of IL-4 ((36.g1±7.87) pg/ml vs. (43.70±6.42)pg/ml, P ＞0.05) and IL-5 ((41.97±7.93) pg/ml vs. (60.14±8.35) pg/ml, P ＜0.05) in BAL fluid and serum slgE ((0.42±0.05)vs. (0.75±0.06) OD units, P ＜0.05) were profoundly reduced. However, the IL-10 level in BAL fluid was significantly increased ((67.74±6.57) pg/ml vs. (44.62±8.81) pg/ml, P ＜0.05).Conclusions These results indicated that 1,25(OH)2D3 pretreatment enhanced the inhibitory effects of immunotherapy on allergic airway inflammation. In the treatment of allergic diseases, 1,25(OH)2D3 pretreatment may be beneficial for improving the efficacy of immunotherapy.