This study investigated the effects of glutamine (GLN) administration on regulating lung γδ T cells in polymicrobial sepsis. Mice were randomly assigned to normal group (NC), septic saline group (SS), and septic GLN group (SG). All mice were fed with chow diet. Sepsis was induced by cecal ligation and puncture (CLP). The SS and SG groups were, respectively, injected with saline and 0.75 g GLN/kg body weight once via tail vein 1 h after CLP. Mice were killed 12 and 24 h after CLP. Their lungs were collected for further analysis. The results showed that, compared with normal mice, sepsis resulted in higher lung γδ T cell and neutrophil percentages and higher cytokine expressed by γδ T cells. Histopathologic findings showed that the extent of inflammatory lesions of the lung alveolar was less severe in the SG group than the SS group after CLP. The SG group had a higher γδ T cell percentage and lower γδ T cell apoptotic rates as well as lower neutrophil numbers in the lungs. Also, interleukin 17A (IL-17A), interferon γ, and IL-10 expressed by γδ T cells and CXC receptor 2 expressed by neutrophils decreased in the SG group. Moreover, GLN reduced IL-17A, IL-1β, and IL-23 concentrations and myeloperoxidase activity in lung tissues. Our results suggest that GLN administration after initiation of sepsis affects lung γδ T cell percentage and cytokine secretion and prevented apoptosis of γδ T cells and neutrophil infiltration to the lungs, which may partly be responsible for ameliorating acute lung injury induced by sepsis.