1,3-β-Glucan was a major cell wall component of fungus. The existing studies showed that 1,3-β-glucan exposure could induce lung inflammation that involved both Th1 and Th2 cytokines. Regulatory T cells (Treg cells) played a critical role in regulating immune homeostasis by adjusting the Th1/Th2 balance. The role of Treg cells and regulatory mechanism in 1,3-β-glucan-induced lung inflammation is still unclear. In our study, mice were exposed to 1,3-β-glucan by intratracheal instillation. To investigate the role of Treg cells in response to 1,3-β-glucan, we generated Treg-depleted mice by intraperitoneal administration of anti-CD25 mAb. The Treg-depleted mice showed more inflammatory cells and severer pathological inflammatory change in lung tissue. Depletion of Treg cells led to increased Th1 cytokines and decreased Th2 cytokines. Treg-depleted mice showed a decreased expression of anti-inflammation cytokine and lower-level expression of CTLA-4. In all, our study indicated that Treg cells participated in regulating the 1,3-β-glucan-induced lung inflammation. Depletion of Treg cells aggravated the 1,3-β-glucan-induced lung inflammation, regulated the Th1/Th2 balance by enhancing Th1 response. Treg cells exerted their modulation function depending on both direct and indirect mechanism during the 1,3-β-glucan-induced lung inflammation.