As it is known to all that the laying rate of commercial laying hens is significantly decreased during the late laying period, paralleled with increased feed conversion rate (FCR) and declined egg quality. However, recent studies demonstrate that reproductive performance as well as egg quality in aged hens can be largely improved by feed supplements. Among them, glycerol monolaurate (GML), a monoglyceride of medium chain fatty acids, is regarded as a promising supplement in poultry production. The aim of this experiment was to evaluate the effect of dietary GML on laying performance, serum biochemical indices, egg quality and gut microbiota in hens during 40–64 weeks of age. A total of 378 (40-week-old) Hy-Line Brown laying hens were randomly assigned into three treatments (21 hens/replicate and 6 replicates/treatment). The control group received a basal diet, and the treated groups fed basal diets containing 150 and 300 mg/kg GML. The results revealed that laying rate (p < 0.05) was significantly increased and the FCR (p < 0.05) was reduced in GML-containing groups compared with the control group. The egg quality (including eggshell thickness and strength) was notably improved by GML supplementation at the later late laying period, and the content of total amino acids and flavor amino acids in the albumen were also increased. Inclusion of 300 mg/kg GML in diet increased the levels of laying-related sex hormones (serum follicle stimulating hormone, luteinizing hormone and estradiol, p < 0.05), serum calcium and lipid (total serum cholesterol, triglycerides and low-density lipoprotein cholesterol, p < 0.05). Moreover, the community and microbial function of gut microbiota were notably altered by GML supplementation. These findings revealed that dietary GML improved the reproductive performance and egg quality mainly by stimulating the sex hormones secretion, serum calcium metabolism and nutrients utilization, which provided us a new approach to simultaneously promote the egg production and improve the egg quality at aged hens.