Background <p>Cold stress has important effects on the growth and production of broiler chickens. Glutamine (<b>Gln</b>) is a conditionally essential amino acid that plays an important role in promoting intestinal development and enhancing immune function. The aim of this study was to investigate the effects of Gln supplementation on the growth performance and health of cold-stressed prestarter broiler chicks.</p> Methods and results <p>A total of 375 1-day-old male SZ901 broilers were randomly divided into five groups (CON, CS, GLN1, GLN2, GLN3). Birds in the CON and CS groups were provided with normal drinking water, while the GLN1, GLN2 and GLN3 groups were provided with water supplemented with 0.4%, 0.8% and 1.2% Gln, respectively. At d7, birds in groups CS, GLN1, GLN2, and GLN3 were stressed at 12 ± 1 ℃ for 12&#xa0;h. The results showed that cold stress significantly decreased the growth performance, serum antioxidant capacity and antibody concentrations, small intestine villus structure, and increased the gene expression of intestinal inflammatory factors of broiler chicks compared with the CON group (<i>P</i> &lt; 0.05). Compared with the CS group, Gln supplementation exhibited increased growth performance, serum antioxidant capacity and antibody concentrations, gene expression levels of intestinal tight junction protein, villus height and villus height to crypt depth ratio (<b>V/C</b>) of small intestine, and decreased mRNA expression level of intestinal inflammatory factors (<i>P</i> &lt; 0.05).</p> Conclusions <p>Gln supplementation ameliorated the impact of cold stress to a large extent as it promoted the development of the intestine and immune system and enhanced the antioxidant enzyme system in cold-stressed prestarter chicks.</p>