<jats:title>Abstract</jats:title><jats:p>Our previous study showed that intraperitoneal injection of α‐galactosylceramide (α‐GalCer) has the ability to activate lung <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cells, but α‐GalCer‐activated <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cells do not result in airway inflammation in wild‐type (<jats:styled-content style="fixed-case">WT</jats:styled-content>) mice. Many studies showed that <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cells had the capacity to induce Treg cells, which gave rise to peripheral tolerance. Therefore, we examined the influence of intraperitoneal administration of α‐GalCer on the expansion and suppressive activity of lung Treg cells using <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cell‐knockout mice and co‐culture experiments in vitro. We also compared airway inflammation and airway hyperresponsiveness (<jats:styled-content style="fixed-case">AHR</jats:styled-content>) after α‐GalCer administration in specific anti‐<jats:styled-content style="fixed-case">CD</jats:styled-content>25 <jats:styled-content style="fixed-case">mA</jats:styled-content>b‐treated mice. Our data showed that intraperitoneal injection of α‐GalCer could promote the expansion of lung Treg cells in <jats:styled-content style="fixed-case">WT</jats:styled-content> mice, but not in <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cell‐knockout mice. However, α‐GalCer administration could not boost suppressive activity of Treg cells in <jats:styled-content style="fixed-case">WT</jats:styled-content> mice and <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cell‐knockout mice. Interestingly, functional inactivation of Treg cells could induce airway inflammation and <jats:styled-content style="fixed-case">AHR</jats:styled-content> in <jats:styled-content style="fixed-case">WT</jats:styled-content> mice treated with α‐GalCer. Furthermore, α‐GalCer administration could enhance <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cells to secrete <jats:styled-content style="fixed-case">IL</jats:styled-content>‐2, and neutralization of <jats:styled-content style="fixed-case">IL</jats:styled-content>‐2 reduced the expansion of Treg cells in vivo and in vitro. Thus, intraperitoneal administration of α‐GalCer can induce the generation of lung Treg cells in mice through the release of <jats:styled-content style="fixed-case">IL</jats:styled-content>‐2 by the activated <jats:styled-content style="fixed-case">iNKT</jats:styled-content> cells.</jats:p>