Coix seed (CS) is widely used as food material and herbal medicine in Asian countries with hypolipidemic and anti-inflammatory properties. But whether CS takes effect by modulating the composition of the gut microbiota remains unknown. Here, three groups of mice were fed different diets for 5 weeks: standard chow, high fat (HF), and CS added to HF. As compared to chow, mice in HF group demonstrated a significant increase in body weight (BW), fat mass (FM), together with total cholesterol (TC), and they even developed impaired glucose tolerance. These HF-mediated deleterious metabolic effects were counteracted partly by complementing CS. 16S rRNA gene sequencing analysis revealed CS increased the abundance of genera Lactobacillus, Coprococcus, and Akkermansia in the gut microbita, and it also enriched species Akkermansia muciniphila and Lactobacillus agilis. A. muciniphila was reported to be inversely associated with obesity, diabetes and cardiometabolic diseases, while L. agilis was negatively associated with TC, BW, FM and blood glucose in our data. We identified CS-altered microbial metabolic pathways that were linked to Glycerolipid metabolism, Biosynthesis of unsaturated fatty acids, sulfur reduction, and glutathione transport system. Our results indicate CS may be used as prebiotic agents to lose weight and prevent obesity-related metabolic disorders.