OBJECTIVE: To study the effects of early intervention on CD4+CD25+ regulatory T cells and TLR4 expression with Mycobacterium phlei F.U.36 in asthmatic mice. METHODS: Thirty female BALB/c mice were randomly divided into three groups: control, asthma model and Mycobacterium phlei F.U.36 treated asthma groups. Asthma was induced by sensitization and challenges with ovalbumin (OVA) in the later two groups. Mycobacterium phlei F.U.36 was intraperitoneally injected 2 weeks before the first sensitization (0.57 μg/time, once every other day for three times) in the intervention group. After 24 hrs of the last challenge, the mice were sacrificed and the left lung tissues were obtained for the observation of lung pathological changes. Splenic mononuclear cells were isolated. The percentage of CD4+CD25+ regulatory T cells in CD4+ T cells and the mean fluorescence intensity of TLR4 on CD4+ CD25+ T cells were detected by flow cytometry. RESULTS: The percentage of CD4+CD25+ regulatory T cells in the asthma model group was significantly lower than that in the control group (P<0.01), but the mean fluorescence intensity of TLR4 on CD4+CD25+ regulatory T cells was not significantly different from the control group. The percentage of CD4+CD25+ regulatory T cells and the mean fluorescence intensity of TLR4 on CD4+CD25+ regulatory T cells increased significantly in asthmatic mice receiving Mycobacterium phlei F.U.36 treatment compared with the asthma group (P<0.01). CONCLUSIONS: Early intervention with Mycobacterium phlei F.U.36 can increase TLR4 expression on CD4+CD25+ cells and the number of CD4+CD25+ regulatory T cells, and thus provides therapeutic effects in asthmatic mice.