Abstract: In order to explore the key metabolites of Bifidobacterium animalis spp. lactis BB-12 metabolizing polysaccharides of Poria cocos, a non-targeted metabolomics method based on liquid chromatography-mass spectrometry (LC-MS/MS) was used, and glucose was used as a control. The effects of polysaccharides of Poria cocos on key metabolites of Bifidobacterium animalis spp. lactis BB-12 were analyzed. The results showed that polysaccharides of Poria cocos could significantly promote the growth of Bifidobacterium BB-12. By using component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), some representative differential metabolites were screened out such as palmatine and ganolucidic acid, etc. There were 12 kinds of key differential metabolites, and 17 differential metabolic pathways were significantly enriched such as ABC transporters, linoleic acid metabolism, and galactose metabolism. These metabolites and pathways demonstrated that Bifidobacterium BB-12 exhibited better growth performance and prebiotic efficacy in the case of fermenting polysaccharides of Poria cocos. This experiment analyzed the metabolites produced by polysaccharides of Poria cocos from the level of small molecular metabolites which were metabolized by Bifidobacterium animalis spp. lactis BB-12. The results provide the theoretical basis and guidance for further study on the probiotic effect of polysaccharides of Poria cocos and Bifidobacterium animalis spp. lactis BB-12 in human bodies.