Presystemic interactions with gut microbiota might play important roles in the holistic action of herbal medicines in their traditional oral applications. However, research interests usually focus on biologic activities of the in vivo available herb-derived components and their exposure in circulation. In this study, we illustrated the importance of studying the presystemic interplay with gut microbiota for understanding the holistic actions of medicinal herbs by using calycosin-7-O-β-D-glucoside (C7G), the most abundant flavonoid and chemical marker in Astragali Radix, as a model compound. When C7G was orally administrated to rats, calycosin-3′-O-glucuronide (G2) was the major circulating component in the blood together with a minor calycosin but not C7G. Rat gut microbiota hydrolyzed C7G in vitro rapidly and produced its aglycone calycosin. Calycosin exhibited higher permeability than C7G and further underwent extensive glucuronidation to yield 3′-glucuronide as the dominant metabolite. Bioactivity assays revealed that G2 exhibited similar or more potent proangiogenic effects than calycosin in human umbilical vein endothelial cells in vitro and in the vascular endothelial growth factor receptor tyrosine kinase inhibitor II-induced blood vessel loss model in zebrafish. More interestingly, the incubation of C7G with gut microbiota from both normal and colitic rats showed a probiotics-like effect through stimulating the growth of the beneficial bacteria Lactobacillus and Bifidobacterium. In conclusion, C7G interacts reciprocally with gut microbiota after oral dosing, which makes it not only an angiogenic prodrug but also a modulator of gut microbiota.