Animal gut is a dynamic ecosystem, and there are many factors affecting the structure of intestinal flora and the diversity of intestinal microbes, among which food differences are the direct causes. To investigate the effect of food composition on the intestinal flora structure of mandarin duck during the reproductive period, 9 feces were collected from 2 foraging sites of Shiqian Mandarin Duck Lake National Wetland Park, Guizhou, China. We analyzed the chloroplast rbcL gene, mitochondrial COI gene and 16S rRNA gene from the total DNA of the feces in detail to know the plants and animals in food compositions and intestinal bacteria of mandarin duck by high-throughput sequencing technology. We found that the gut microbiota composition were significantly correlated with the number of feeding species, the mandarin duck with a wide variety of feeding food had the more complex gut flora with more probiotics and less pathogenic bacterium. This study provides further theoretical support for food difference to affect the changes of intestinal bacterial structure of host, and provides scientific basis for understanding the feeding preference of mandarin duck at breeding area.