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Ginseng polysaccharides increase the hypoglycemic activity of ginsenoside Rb1.Ginseng polysaccharides alter the biotransformation pathway of ginsenoside Rb1.Ginseng polysaccharides enhance the biotransformation rate of ginsenoside Rb1 into CK.This is the first study to show the synergistic effect of ginsenoside Rb1 and ginseng polysaccharides for diabetic treatment.Ginseng polysaccharides (GP) have been reported to modulate gut microbiota, and ginsenoside Rb1 is known to display significant hypoglycemic activity. However, the synergistic effect of Rb1 and GP when applied to diabetic treatment remains largely unknown. Male rats were divided into ten groups: blank group (B-Group), model group (D-Group), Rb1group (Rb1-Group), CK group (CK-Group), GP groups and GP+Rb1groups in dosage of high, middle and low (H-Group, M-Group, L-Group, H-Rb1-Group, M-Rb1-Group, and L-Rb1-Group). CK-Group, GP groups and Rb1group were fed CK, GP and Rb1 for 30 days, respectively. GP+Rb1groups were fed GP on the initial 15 days and GP and Rb1 on the final 15 days. The fasting glucose of all groups was measured every five days. The transformation of Rb1 in vitro by rat intestinal microflora, which was collected from the B-Group, D-Group and GP groups on the 15th day, was investigated using HPLC and RRLC-Q-TOF/MS. Analyses the of 16S rRNA gene of the fecal bacterial population and fecal β-glucosidase activity were conducted among the B-Group, D-Group and H-Group. Compared with those of rats in the D-Group, the fasting glucose levels of rats in the CK-Group and H-Rb1-Group decreased highest. During transformation of Rb1 by diabetic rat intestinal microflora, five transformed products, including ginsenoside Rd, F2, CK, gypenoside XVII (G-XVII), and LXXV (G-LXXV), as well as three transformation pathways, were identified. When a high dose of GP was fed to diabetic rats for 15 days, the formation of intermediates, including G-XVII and G-LXXV was inhibited, and only one pathway (Rb1→Rd→F2→CK) was identified. Moreover, the biotransformation rate of CK increased from 14.0% to 86.7% after 8h of cultivation. GP reinstated the perturbed holistic gut microbiota and promoted fecal β-d-glucosidase activity. Ginsenoside Rb1 and GP shows synergistic effects when applied to diabetic treatment and may be developed as a potential antidiabetic drug.