This study preliminarily investigated the mechanism by which chloroquine (CQ) relieves acute lung injury (ALI) complicated in acute hemorrhagic necrotizing pancreatitis (AHNP). Sixty male Wistar rats were randomized into sham-operated group (group A, n=10), AHNP group (group B, n=10), L-arginine-treated group (group C, n=10), L-N-nitro-L-arginine methyl ester (NAME)-treated group (group D, n=10), CQ-treated group (group E, n=10) and CQ+L-NAME-treated group (group F, n=10). TLR4 expression was measured by using real time-PCR and Western blotting respectively. The results showed that, in the group B, the expression of TLR4 and the levels of TNF-α and IL-6 in the lungs were significantly increased, and the nitric oxide (NO) concentration was reduced, as compared with those in the group A (P<0.05 or P<0.01). Lung injury was aggravated with the increased expression of TLR4. When the inhibitor and stimulator of TLR4, namely L-Arg and L-NAME, were added respectively, lung injury was correspondingly relieved or aggravated (P<0.05 or P<0.01). In the group E, TLR4 expression was substantially lower and NO concentration higher than those in the group B (P<0.05 or P<0.01). However, in the group F, NO concentration was markedly decreased, and the inhibitory effect of CQ on TLR4 expression and the relief of lung injury were weakened when compared with those in the group E (P<0.05 or P<0.01). It was concluded that TLR4 may play an important role in the pathogenesis and development of ALI complicated in AHNP. CQ could relieve ALI by decreasing the TLR4 expression and increasing the NO release.