Objective To investigate the pathogenesis of paraquat (PQ) induced acute lung injury through Toll like receptor 3 (Toll-like receptor-3, TLR3), TLR induced nuclear transcription factors (Nuclear Factor-kappa B, NF-κB) and its downstream pro-inflammatory factors TNF-a, IL-1β, IL-6. Methods The acute lung injury model of mice and the acute injury model of type II alveolar epithelial cells (A549) induced by PQ were established. The PQ mediated pathological changes of lung tissue, the cell count and cytospin of bronchoalveolar lavage fluid (BALF) were evaluated, and the pro-inflammatory factors in the lung tissue of mice were determined by ELISA the viability of A549 cells mediated by PQ was detected by CCK8 assay, and the mRNA expression and protein level of TLR3, Phospho-NF-kBp65, tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in the lung tissues and A549 cells were observed by Real-time PCR and Western-blotting. In control group, the mice received normal saline (NS) instead of PQ. Results Compared with the control group, the mice in PQ group showed difficulty breathing, decreased activity, reduced food intake, and weight lost. The total number of BALF cells in the PQ group was significantly increased [NS: (0.018 8±0.102 1) × 105 vs. PQ: (0.237 4±0.121 7) ×105,t=9.804,P＜0.01] with macrophage [NS: (0.162 8±0.086 5) × 105 vs. PQ: (1.063 3± 0.343 3) × 105,t=8.043,P＜0.01],lymphocyte [NS: (0.006 6±0.005 2) × 105 vs. PQ: (0.171 2±0.099 1) × 105, t=5.243,P＜O.Ol] and neutrophils [NS: (0.000 04±0.000 1) × 105 vs. PQ: (0.901 9±0.652 5) × 105, t=4370, P＜0.01]. In PQ group, the appearance and volume of lung tissue increased with hyperemia and edema. HE slices showed inflammatory cell infiltration and pulmonary interstitial hemorrhage. Moreover, the expressions of TNF-a, IL-1β, IL-6 in BALF of PQ group was significantly higher than those of the control group by the ELISA assay [TNF-a: NS: (2.782 1 ± 3.521 5) vs. PQ: ( 7.512 6±3.459 8) pg/mL,t=3.030, P＜0.05; IL-1β: NS: (22.687 5±14.229 3) vs. PQ: (163.100 4±81.118 3) pg/mL,t=5.391,P＜0.01 ; IL-6: NS: (1.653 3±0.442 7) vs. PQ: (648.565 6 ± 422.606 1) pg/mL, t=4.841,P＜0.01]. CCK8 results indicated that the viability of A549 cells decreased by 25.3％ and 36.4％ at 24h after 200～400 μmol/L PQ treatment (all P＜0.05). The mRNA expressions of TNF-a, IL-1p, IL-6 in the lung tissue and A549 cells in PQ group were higher than those in the control group as well (all P＜ 0.05). Furthermore, Western-blotting results revealed that the protein levels of TLR3 and Phospho-NF-κBp65 in the lung and A549 cells mediated by PQ were significantly higher than those in the control group (all P＜ 0.05). Conclusions PQ may induce acute lung injury by up-regulation of the expressions of inflammatory factors TNF-a, IL-1β, IL-6 through the TLR3/NF-κB signaling pathway.