At present, the timing of glutamine administration remains controversial. The aim of the present study was to confirm the early protective mechanisms of alanyl‑glutamine (Ala‑Gln) against lipopolysaccharide (LPS)‑induced lung injury, as well as to detect the best time for Ala‑Gln usage. A total of 60 adult Wistar rats were randomly divided into 6groups: The control group (C), the LPS‑induced shock group (LPS), the pre‑Ala‑Gln treated group (A1) and the pre‑Gln treated group (G1), which separately received 4.5% Dipeptiven and 3% glutamine just before LPS administration; the post‑Ala‑Gln treated group (A2) and the post‑Gln treated group (G2), which was respectively infused with 4.5% Dipeptiven and 3% glutamine at 1h following LPS. Survival rates were observed at 6h following the LPS injection. Blood samples were drawn for analysis of cytokine levels 1h prior to (T0) and 6h following (T1) LPS injection. All rats were killed at T1 and the pulmonary samples were collected. Plasma concentrations of tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑8 at T0 and T1, apoptosis in lung epithelial cells and the expression of heat shock protein (HSP)70 were detected. The lung wet/dry weight ratio (W/D) and the content of protein in the bronchoalveolar lavage fluid (BALF) were also determined. Survival rates at 6h following (T1) LPS administration were both 100% in groups A1 and G2, but 70% in A2 and G2 groups. The W/D, the content of protein in BALF and cytokine levels were significantly lower in groups A1 and G1 than that in group LPS (P<0.05) at T1. The apoptosis index of both alveolar and bronchial epithelial cells was obviously lower in A1 and G1 groups than that in the LPS group (P<0.05). Gray gradients of HSP70 in the A1 and G1 groups were dramatically higher than those of group LPS (P<0.05). In conclusion, pre‑administration of Ala‑Gln just before LPS can effectively protect the lung by enhancing HSP70 expression, but delayed administration cannot protect LPS‑induced lung injury.