Background Subcutaneous specific immunotherapy has been demonstrated to be capable of inducing T-cell regulatory response. Interleukin-10 (IL-10) plays a crucial role in inducing allergen-specific tolerance. However the reports of the changes of IL-10 in house dust mite (HDM)-specific immunotherapy were varied. The aim of this study was to evaluate the function of IL-10-secreting regulatory T cells in asthma children successfully treated with HDM immunotherapy. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 27 patients following 1.5-2 years of HDM-specific immunotherapy (SIT, SIT group) and from 27 matched treated asthmatic children allergic to HDM (asthma group). After 48 hours of in vitro stimulation with HDM extracts, IL-10-secreting regulatory T cells were measured by four colour flow cytometry. Sera were tested for allergen-specific IgG(4) and IgE using the Immuno CAP 100 assay. Results PBMCs from children undergoing immunotherapy following HDM extracts stimuli produced significantly more IL-10 compared with the asthma group. The frequency of iTreg cells and aTreg cells increased in SIT group after HDM stimulation, while it was not affected in the asthma group. Among the iTreg cells and aTreg cells, the frequency of CD4(+)CD25(-)Foxp3(-)IL-10(+) Treg cells increased the most which was 2 times higher than that in unstimulated cultures in SIT group. The levels of HDM-specific IgG4 of SIT group was significiently higher compared with asthma group, but there was no correlation of the levels of HDM-specific IgG4 and IL-10 secreting Treg cells. Conclusions HDM-specific immunotherapy can successfully upregulate the frequency of IL-10-secreting Treg cells. CD4(+)CD25(-)Foxp3(-)IL-10(+) Treg cells may play a key role in inducing the immune tolerance in HDM-specific immunotherapy. Chin Med J 2010;123(15):2099-2104