Glycerol monolaurate (GML) has potent antimicrobial and anti-inflammatory activities. The present study aimed to assess the dose-dependent antimicrobial-effects of GML on the gut microbiota, glucose and lipid metabolism and inflammatory response in C57BL/6 mice. Mice were fed on diets supplemented with GML at dose of 400, 800 and 1600 mg kg−1 for 4 months, respectively. Results showed that supplementation of GML, regardless of the dosages, induced modest body weight gain without affecting epididymal/brown fat pad, lipid profiles and glycemic markers. A high dose of GML (1600 mg kg−1) showed positive impacts on the anti-inflammatory TGF-β1 and IL-22. GML modulated the indigenous microbiota in a dose-dependent manner. It was found that 400 and 800 mg kg−1 GML improved the richness of Barnesiella, whereas a high dosage of GML (1600 mg kg−1) significantly increased the relative abundances of Clostridium XIVa, Oscillibacter and Parasutterella. The present work indicated that GML could upregulate the favorable microbial taxa without inducing systemic inflammation and dysfunction of glucose and lipid metabolism.