The permeation and efflux of six polysubstituted flavonoids isolated from Crossostephium chinense, a Chinese traditional and herbal drug for the treatment of diabetes, were investigated using the Caco-2 cell monolayer. The six flavonoids (selagin, apometzgerin, tricetin-3′,4′,5′- trimethylether, quercetagetin-3,6,7-trimethylether, hispidulin and quercetagetin) sharing a similar parent skeleton structure with varied substituents in the heterocyclic ring B were selected for the study. Quercetagetin exhibited a low bi-directional permeability comparable to that of atenolol, suggesting a paracellular diffusion mechanism. The remaining compounds exhibited time- and concentration- dependent permeation with apparent permeability coefficient (P) values in the range 10 -10 cm/s, suggesting transcellular diffusion pathways. Selagin exhibited significantly larger basolateral to apical P than that of the reverse direction, suggesting the existence of efflux mechanisms. ATP-depletion and probenecid pretreatment led to a significant inhibition of the efflux of selagin, whereas verapamil had no effect on the basolateral to apical transport of selagin, suggesting that multidrug resistance proteins (MRPs) and not P-glycoprotein play a role in the intestinal efflux of selagin. Based on present and previous results, the structure-permeation relationship and the role of MRPs in mediating the efflux of flavonoids are discussed. Experimental results in this study provide useful information for pharmacological applications of the flavonoids from C. chinense. Copyright © 2010 John Wiley & Sons, Ltd.