Extensive interactions occur between a poultry host and its gut microbiome. Glycerol monolaurate (GML) possesses a large range of antimicrobial and immunoregulatory properties. This study was conducted to investigate the impact of different doses of GML (basal diets complemented with 0, 300, 600, 900, or 1200 mg/kg GML) on growth performance, intestinal barrier, and cecal microbiota in broiler chicks. Results revealed that feed intake increased after 900 and 1200 mg/kg GML were administered during the entire 14-day experiment period. Dietary GML decreased crypt depth and increased the villus height-to-crypt depth ratio of the jejunum. In the serum and jejunum, supplementation with more than 600 mg/kg GML reduced interleukin-1β, tumor necrosis factor-α, and malondialdehyde levels and increased the levels of immunoglobulin G, jejunal mucin 2, total antioxidant capacity, and total superoxide dismutase. GML down-regulated jejunal interleukin-1β and interferon-γ expression and increased the mRNA level of zonula occludens 1 and occludin. A reduced expression of toll-like receptor 4 and a tendency of down-regulated nuclear factor kappa-B was shown in GML-treated groups. In addition, GML modulated the composition of the cecal microbiota of the broilers, improved microbial diversity, and increased the abundance of butyrate-producing bacteria. Spearman's correlation analysis revealed that the genera Barnesiella, Coprobacter, Lachnospiraceae, Faecalibacterium, Bacteroides, Odoriacter, and Parabacteroides were related to inflammation and intestinal integrity. In conclusion, GML ameliorated intestinal morphology and barrier function in broiler chicks probably by regulating intestinal immune and antioxidant balance, as well as intestinal microbiota.