The immunomodulatory effects of PD-1 and CD4⁺CD25⁺ T_regs in the resolution of ALI are still poorly understood. Accordingly, 1 million T_regs were isolated from spleens of WT C57BL/6 or PD-1^−/− mice (magnetical bead purification and subsequent labeling with/without Vybrant dye) and then AT into mice subjected to Hem shock during their resuscitation period, which were subsequently subjected to CLP/septic challenge (24 h post-Hem) to induce iALI. Initially, we demonstrated that Vybrant-labeled AT T_regs appear in the lungs of iALI mice. Subsequently, we found that AT of WT T_regs induced a significant repression of the indices of lung injury: a reduction of neutrophil influx to the lung tissue and a decrease of lung apoptosis compared with vehicle-treated iALI mice. In addition, these mice had substantially higher concentrations of BALF and lung-tissue IL-10 but significantly decreased levels of lung KC. However, these beneficial effects of the AT of T_regs were lost with the administration of PD-1^−/− mouse T_regs to the recipient WT mice. ALI was exacerbated in these recipient mice receiving AT PD-1^−/− T_regs to the same extent as iALI mice that did not receive T_regs. These data imply that T_regs can act directly to modify the innate immune response induced by experimental iALI, and this is mediated, in part, by PD-1. Hence, the manipulation of T_regs may represent a plausible target for treating iALI.