Heat shock protein 70 (HSP70) is reported as the main factor responsible for the beneficial effects of glutamine (GLN) and as a negative regulator of high mobility group box protein-1 (HMGB-1) expression. Our aim was to determine whether GLN attenuates acute lung injury (ALI) by the inhibition of HMGB-1 expression during sepsis. Male Sprague–Dawley rats were subjected to caecal ligation and puncture (CLP) to induce sepsis. GLN or saline was administered through tail vein 1 h after CLP. Then, quercetin (Q), an inhibitor of HSP70, was utilised to assess the role of the enhanced HSP70. We observed the survival of the subjects. At 24 h post-CLP, we measured lung HSP70, phosphorylated heat shock factor-1 (HSF-1-p) and HMGB-1 expressions, NF-κB DNA-binding activity and ALI occurrence. We also measured serum HSP70, IL-6 and HMGB-1 concentrations. GLN improved survival during sepsis. In GLN-treated rats, lung HSP70 and HSF-1-p expressions were enhanced, lung HMGB-1 expression and NF-κB DNA-binding activity were suppressed, and ALI was attenuated. Furthermore, in GLN-administered rats, serum HSP70 concentration was higher, and serum IL-6 and HMGB-1 concentrations were lower than those in non-treated rats. Q inhibited the enhancement of HSP70 and HSF-1-p expressions and abrogated the GLN-mediated benefits. In conclusion, GLN attenuated ALI and improved survival by the inhibition of HMGB-1 expression during sepsis in rats. These benefits were associated with the enhancement of HSP70 expression by GLN.