Personalized diets change the internal metabolism of organisms, which, in turn, affects the health of the body; this study was performed to explore the regulatory effects of polysaccharides extracted from Auricularia auricula on the overall metabolism and gut microbiota in normal C57BL/6J mice. The study was conducted using metabolomic and microbiomic methods to provide a scientific basis for further development and use of Auricularia auricula resources in the Qinba Mountains and in nutritional food with Auricularia auricula polysaccharides (AAP) as the main functional component. Based on LC-MS/MS metabolomic results, 51 AAP-regulated metabolites were found, mainly enriched in the arginine biosynthesis pathway, which had the highest correlation, followed by the following metabolisms: arginine and proline; glycine, serine and threonine; and glycerophospholipid, along with the sphingolipid metabolism pathway. Furthermore, supplementation of AAP significantly changed the composition of the mice intestinal flora. The relative abundance levels of Lactobacillus johnsonii, Weissella cibaria, Kosakonia cowanii, Enterococcus faecalis, Bifidobacterium animalis and Bacteroides uniformis were markedly up-regulated, while the relative abundance of Firmicutes bacterium M10-2 was down-regulated. The bioactivities of AAP may be related to the regulatory effects of endogenous metabolism and gut microbiota composition.