Efflux transporters are extensively distributed and expressed in the intestinal epithelium and contribute to the low oral bioavailability of flavonoids and flavonoid glucuronides by pumping these compounds back into intestinal lumen. Our previous study has shown the inhibitory effect of Cremophor EL, a non-ionic surfactant, on efflux transporter multidrug resistance-associated protein (MRP) 2. In the current study, by using membranes overexpressing several common ATP-binding cassette (ABC) transporters including P-glycoprotein (P-gp), MRP1, MRP2, MRP3 and breast cancer resistance protein (BCRP), scutellarin, a poorly water-soluble flavonoid, was identified as the substrate of MPR2, MRP3 and BCRP. The effects of Cremophor EL on the transmembrane transportation of scutellarin by MRP2, BCRP, and MRP3 were investigated with inside-out Sf9 vesicles. Results showed that at nontoxic concentrations, Cremophor EL enhanced the transportation of scutellarin by MRP3 and inhibited the efflux transportation of scutellarin by MRP2 and BCRP concurrently. The relations between Cremophor EL and these transporters were explored using MDCK II-MRP2, MDCK II-BCRP, and MDCK II-MRP3 cell models. Compared with the control group, 5 μg/ml Cremophor EL decreased the Papp(BL-AP) of scutellarin in MDCK II-MRP2 cell monolayers by > 4 fold (from 13.57 ± 0.76 × 10− 7 to 2.90 ± 0.14 × 10− 7 cm/s), and the Papp(BL-AP) in MDCK II-BCRP cell monolayers decreased from 9.12 ± 0.15 × 10− 7 to 6.34 ± 0.08 × 10− 7 cm/s. On MDCK II-MRP3 cell monolayers, 5 μg/ml Cremophor EL increased the Papp(AP-BL) of scutellarin by 3.5 fold (from 7.88 ± 0.43 × 10− 7 to 2.79 ± 1.61 × 10− 6 cm/s), and caused an over 5-fold increase in Papp(AP-BL)/Papp(BL-AP). These findings suggested that Cremophor EL possesses the potent ability of inhibiting MRP2 and BCRP, as well as activating MRP3 effectively. In vivo pharmacokinetic research in rats further confirmed the improvement of oral absorption of scutellarin by Cremophor EL. In summary, our present study has identified a new mechanism for increasing the oral absorption and bioavailability of poorly absorbed drugs in which Cremophor EL increased MRP3 mediated transport but reduced MRP2 and BCRP mediated efflux concurrently, thereby enhancing the entry of drugs from enterocytes into the blood circulation.