Allergic asthma is a chronic inflammatory disease and despite the introduction of potent and effective drugs, the prevalence has increased substantially over the past few decades 1. The explanation that has attracted the most attention is the'hygiene hypothesis', which suggests that the increase in allergic diseases is caused by a cleaner environment and fewer childhood infections 2, 3, 4. Indeed, certain mycobacterial strains can cause a shift from T-helper cell 2 (Th2) to Th1 immune responses, which may subsequently prevent the development of allergy in mice 5, 6, 7. Although the reconstitution of the balance between Th1 and Th2 is an attractive theory, it is unlikely to explain the whole story, as autoimmune diseases characterized by Th1 responses can also benefit from treatment with mycobacteria and their prevalence has also increased in parallel to allergies 8. Here we show that treatment of mice with SRP299, a killed Mycobacterium vaccae-suspension, gives rise to allergen-specific CD4+ CD45RB Lo regulatory T cells, which confer protection against airway inflammation. This specific inhibition was mediated through interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), as antibodies against IL-10 and TGF-β completely reversed the inhibitory effect of CD4+ CD45RB Lo T cells. Thus, regulatory T cells generated by mycobacteria treatment may have an essential role in restoring the balance of the immune system to prevent and treat allergic diseases.