The effects of glycerol monolaurate （GML） on the glucose and lipid metabolism， systemic inflammatory response and the time-dependent influence on the gut microbiota in C57BL/6 mice were investigated. Results showed that GML neither affected lipid profiles and glycemic markers nor induced chronic systemic inflammation. However， GML promoted the growth of mice significantly and decreased the ratio of low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol at a trend level. High-throughput sequencing showed that GML reduced the Shannon and Simpson diversity index after 5-week and 17-week treatment. At the phylum level， GML changed the microbial compositions significantly after 5-week treatment， but had no such effect after 17-week treatment. At the genus level， the time-dependent effect on the microbiota within the control group （NCD） was greater than that on the GML-treated group （G1200）， thus the differential taxa between NCD and G1200 group at 5 weeks and 17 weeks were different. However， the promoting effect of GML on the beneficial taxa， such as Barnesiella， Oscillibacter and Clostridium XIVb， and inhibitory effect on the pathogenic Anaeroplasma precede the time-dependent effect， indicated that GML could not only maintain the stability of the microbiota， but also optimized the microbial structure. Furthermore， GML could efficiently stabilize the mucosal barrier by significantly increasing the mRNA levels of intestinal α-defensin and occludin， which were correlated with the beneficial taxa mediated by GML.