Glutamine (GLN) has been shown to protect against inflammatory injury and illness in experimental and clinical settings. The mechanism of this protection is unknown, however, laboratory and clinical trial data have indicated a relationship between GLN-mediated protection and enhanced heat shock protein 70 (HSP70) expression. The aim of this study was to examine the hypothesis that GLN's beneficial effect on survival, tissue injury, and inflammatory response following inflammatory injury is dependent on HSP70 expression. Mice with a specific deletion of the HSP70 gene underwent cecal-ligation and puncture (CLP) induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo one-hour post-CLP. Lung tissue NF-kB activation, inflammatory cytokine response, and lung injury were assessed post-CLP. Survival was assessed for 5 days post-CLP. Our results indicate that GLN administration improved survival in Hsp70^+/+ mice versus Hsp70^+/+ mice not receiving GLN, however GLN exerted no survival benefit in Hsp70^-/- mice. This was accompanied by a significant decrease in lung injury, attenuation of Nf-kB activation, and pro-inflammatory cytokine expression in GLN treated Hsp70^+/+ mice versus Hsp70^+/+ mice not receiving GLN. In the Hsp70^-/- mice, GLN's attenuation of lung injury, Nf-kB activation, and pro-inflammatory cytokine expression was lost. These results confirm our hypothesis that HSP70 expression is required for GLN's effects on survival, tissue injury, and the inflammatory response following global inflammatory injury.