Epimedium spp. is commonly used in Traditional Chinese Medicine. Epimedins A, B, and C are three major bioactive flavonoids found in Epimedium spp. that share similar chemical structures. In this study, the intestinal absorption mechanism of these three compounds was investigated using the Caco-2 cell monolayer model in both the apical-to-basolateral (A-B) and the basolateral-to-apical (B-A) direction. The absorption permeability (PAB) of epimedins A, B, and C were extremely low and increased as the concentration of the epimedins increased from 5 to 20 μM, but, at 40 μM, the PAB values were reduced. Meanwhile, the amount of transported compounds increased in a time-dependent manner. The PAB of epimedins A and C were significantly increased and efflux ratios decreased in the presence of verapamil (an inhibitor of P-glycoprotein) and dipyridamole (an inhibitor of breast cancer resistance protein) while, in the presence of MK571 (an inhibitor of multidrug resistance proteins), the absorption of epimedins A and C did not change significantly, indicating that P-gp and BCRP might be involved in the transport of epimedins A and C. The PAB of epimedin B significantly increased while its secretory permeability (PBA) significantly decreased in the presence of dipyridamole, indicating that BCRP might be involved in the transport of epimedin B. No obvious changes in the transport of epimedin B were observed in the presence of verapamil and MK571. In summary, our results clearly demonstrate, for the first time, that poor bioavailability of these three prenylated flavonoids is the result of poor intrinsic permeability and efflux by apical efflux transporters.