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Nasal administration is a high-potential delivery system, particularly because it can provide a pathway from the nose to the brain. The objective of this research is to characterize puerarin transport across a Calu-3 cell monolayer used as a model of the nasal mucosa and to evaluate the influence of puerarin in combination with paeoniflorin and menthol to explore the enhanced mechanism of the permeability at the cell level. The apparent permeability coefficients (Papp) of puerarin bidirectional transport were both <1.5×10−6 cm/s, and the efflux ratio was <1.5, indicating that puerarin alone exhibited poor absorption and that its transport primarily occurred by passive diffusion through the cell monolayer. When puerarin was coad ministered with paeoniflorin, the Papp was not changed (P>0.05). However, the addition of menthol significantly (P<0.05) improved the Papp of puerarin in both directions. Moreover, based on immunofluorescence experiments and transepithelial electrical resistance measurements, the data indicated that the drug compatibility opened tight junctions and weakened the barrier capabilities of epithelial cells, thereby promoting the permeability of puerarin.