In order to investigate the effects of different terms of inhaled nitric oxide (NO) preconditioning with low concentration on the activations of Toll-like receptor 2 and 4 (TLR2/4) in the lung ischemia-reperfusion (IR) injury in mice, we divided the male C57BL mice into five groups: sham (S) group, IR group, NO 1-min preconditioning group (15 ppm NO inhalation for 1 min before ischemia, NO 1-min), NO 10-min preconditioning group (15 ppm NO inhalation for 10 min before ischemia, NO 10-min), NO 60-min preconditioning group (15 ppm NO inhalation for 60 min before ischemia, NO 60-min). The changes of partial pressure of oxygen in artery (PaO2), left lung wet-to-dry weight ratio (W/D), and myeloperoxidase (MPO) in the injured lung were measured in every group at 6th h of reperfusion after 60 min of left lung ischemia. The changes of TLR2/4 activations and plasma TNF-α were measured in this procedure in additional mice. As compared with IR group, PaO2 increased, MPO and W/D decreased evidently after reperfusion in NO 10-min group. The changes in NO 60-min group were similar to those in NO 10-min group. There was no difference between NO 1-min and IR group. In NO inhalation group, the expressions levels of TLR2/4 mRNA and proteins were diminished, TNF-α concentrations were decreased, and the lung injuries were ameliorated effectively. We concluded that short term inhalation of NO protected lung IR injury. But the protective effect of NO was not increased with extension of inhaled NO. Inhaled NO could inhibit the activations of TLR2/4 in the lung after IR injury. TLR signal pathway might contribute to the effect of protection with NO in this model.