Acute kidney injury (AKI) is a common problem characterized by an inflammatory response in the kidney and oxidative stress. However, there are no interventions to prevent AKI. Glutamine is an important precursor of glutathione and has also been shown to induce heat shock proteins (HSP). Thus, glutamine may affect both oxidative stress and inflammation. This study was to explore the effects of glutamine pretreatment on nephrotoxic AKI and to investigate the underlying mechanisms. First, the effects of alternate doses of glutamine were compared in CD-1 mice with AKI induced with folic acid intra-peritoneal injection. Then the effects of glutamine quercetin (an HSP inhibitor), and quercetin+glutamine, were compared in the same AKI model. AKI were assessed with plasma creatinine, urine neutrophil gelatinase-associated lipocalin, and renal histology. Inflammatory response was monitored with renal tumor necrosis factor (TNF-α), chemkines (CXCL1 and CCL2) contents, and neutrophil infiltration. Oxidative injury was detected with reduced glutathione, malondialdehyde, and protein thiol. Glutamine provided dose-dependent renal protection. Pretreatment with quercetin, which was showed to inhibit HSP-70 expression, abolished glutamine's renal-protective effects. Quercetin also abrogated glutamine's beneficial effects on renal TNF-α, chemokines, and neutrophil infiltration. However, quercetin did not affect glutamine's anti-oxidative effects. These results suggest that glutamine's renal-protective effects are mainly related to its activation of HSP-70, which mitigates inflammatory response, renal neutrophil infiltration and subsequent AKI. Regulating neutrophil infiltration might be a potential therapeutic target for AKI.