Recent studies reveal that glycerol monolaurate (GML) is regarded as an effective feed supplement in the production of broilers; however, the underlying mechanism remains unknown. The current study aimed to investigate how GML affected production performance and meat quality in yellow-feathered broilers. A total of 528 chicks were randomly assigned into four groups for a 56-day feeding trial. The control group received a basal diet, and the treated groups fed basal diet containing 300 (GML300), 450 (GML450), and 600 (GML600) mg/kg GML. Results revealed that dietary GML notably increased the average daily feed intake (p < 0.05) and body weight in broilers during 28-56 days of age and improved the duodenum and jejunum morphology. Dietary GML increased the total cholesterol in broilers (p < 0.05), but the hepatic, abdominal, and muscular fat deposition, as well as muscle fatty acids, were not affected. The flavor amino acids and total amino acids in muscle of GML300 and GML 450 groups were notably (p < 0.05) increased. GML supplementation selectively increased the colonization of an unclassified genus of Lachnospiraceae family and Bifidobacteriaceae, which were significantly (p < 0.05) correlated with the increase of muscle amino acids. Meanwhile, dietary GML notably increased short chain fatty acids content and the microbial DNA abundance of carbohydrate, amino acids and lipid metabolism pathway in cecum. These findings demonstrated that dietary GML improved performance, intestinal morphology, and muscle amino acids in broilers mainly by manipulating community, function and metabolites of gut microbiota.