Hawthorn leave flavonoids (HLF) are widely used as an herb or dietary supplements for cardio-cerebrovascular diseases. However, its gastrointestinal absorption behavior and mechanism have not been disclosed. In this study, gastrointestinal absorption and its regulation of 4''-O-glucosylvitexin (GLV), 2''-O-rhamnosylvitexin (RHV), vitexin (VIT), rutin (RUT) and hyperoside (HP) in HLF were investigated using in vitro, in situ and in vivo models. Apparent permeability coefficient (P) of five flavonoids were (2.18 ± 0.15) ×10~(3.83 ± 0.22) ×10 cm·sec across the Caco-2 cells. GLV, RHV, VIT and RUT demonstrated similar and poor absorption in rat stomach (absorption percentage per hour (A), (1.78 ± 0.14 ~ 5.69 ± 0.51)%·h) and small intestine (absorption rate ([Formula: see text], (0.012 ± 0.006 ~ 0.055 ± 0.003) h), extent (A, (1.28 ± 0.14 ~ 2.82 ± 0.19)% ·h) and potential (P, (0.31 ± 0.05 ~ 1.41 ± 0.08)×10 cm·sec)), while HP showed relatively better absorption (P, (2.55 ± 0.15 ~ 4.27 ± 0.36)×10 cm·sec) in rat small intestine. Absorption of five flavonoids exhibited dose-dependence, pH-dependence and region-selectivity. Meanwhile, these flavonoids were excreted via intestine, secreted via bile and metabolized by intestinal microflora. Their absorption was significantly increased by absorption enhancers (cow bile salt and sodium dodecyl sulphate), transporter regulators (verapamil hydrochloride, digoxin and rifampicin). GLV and RHV exhibited enterohepatic circulation after oral administration of HLF. In conclusion, flavonoids in HLF were absorbed via passive diffusion accompanied with active transport, intestinal microflora metabolism, bile secretion and intestinal efflux. They belong to BCS class III ("like" drugs with high solubility and low permeability) and displayed poor oral bioavailability.