Abstract: Water in oil (W/O) microemulsion formulation was developed to enhance intestinal absorption of ginsenoside Rb₁ (Rb₁) of panax notoginseng (PNS). Effects of W/O microemulsions on pharmacokinetics after intraduodenal administration, membrane fluidity and membrane transport of Rb₁ were studied in rats, liposomes and parallel artificial membrane permeability assay (PAMPA), respectively. Soybean phospholipids/ethanol (SP/EtOH) was selected as surfactant/cosurfactant, together with PNS 400 mg·mL^-1 solution and various kinds of oils, to prepare 11 W/O microemulsions. Most of the microemulsions can enhance Rb₁ intestinal absorption significantly. Besides surfactant/cosurfactant, oil also had an effect on the enhanced absorption and the order of enhancement was as follows: glyceryl laurate ≈ isopropyl myristate > isopropyl palmitate > 2-ethylhexanol palmitate. The effection of absorption enhancement by the long chain glyceride (>C₁₄) is lower than that by the medium chain glyceride (C₈-C₁₄). Most of W/O microemulsions were found to enhance the membrane fluidity of liposomes to different extents. In PAMPA analysis, efficient permeability coefficient (P_e) of diluted-microemulsion (D-ME) is mostly higher than that of PNS solution, which indicated the components of microemulision can facilitate the membrane permeability of the drug. Meanwhile, linearity correlation between P_e and ratio of relative bioavailability (F_r) was acquired for undiluted microemulison (ME). Therefore, W/O microemulsions can enhance intestinal absorption of Rb₁, and this effect may be attiributed to its enhancement on membrane fluidity to a certain degree. PAMPA analysis could be brought into not only the investigation of membrane transport of crude drug, but also conditioned preformulation research (e.g. absorption enhancer etc.).