Flavonoid glycosides are metabolized by intestinal bacteria, giving rise to a wide range of phenolic acids that may exert systemic effects in the body. The microbial metabolism of flavonoids extracted from the leaves of Diospyros kaki (FLDK) by intestinal bacteria was investigated in vitro. High-performance liquid chromatography/linear trap quadrupole orbitrap mass spectrometry was performed to analyze the metabolites of flavonoids in vivo using Xcalibur2.1 software. The results showed that the levels of flavonoid glycosides and flavonoid aglycones decreased rapidly in the process of microbial metabolism by intestinal bacteria in vitro, and the metabolic rate may be related to the concentration of intestinal bacteria in the culture solution. In vivo metabolites of FLDK were detected in rat plasma and urine after oral administration of FLDK. Eight flavonoids were identified in the urine, and three were identified in the plasma; however, flavonoid aglycones were not found in the plasma.