This study was conducted to investigate the role of P-glycoprotein (P-gp) and Multidrug resistance-associated protein 2(Mrp2) in the rat intestinal absorption of scutellarin and explore the possible reasons for its low oral bioavailability. Verapamil had little effect on the transport amount of scutellarin shown by in vitro everted sac experiments and the apparent permeability of the drug demonstrated by in situ single-pass intestinal perfusion experiments (SPIP). Leukotriene C4 (LTC4) added to the mucosal side significantly enhanced the transport of scutellarin to the serosal side. The Papp value of scutellarin increased gradually on raising the L-Buthionine-[S,R]-sulfoximine (BSO) concentration to 0.5 mM in the perfusion solution (P < 0.05). When probenecid (1 mM) was coperfused, a 1.34-fold increase in the Papp was observed (P < 0.05). Coperfusion of 0.5 mM BSO and 1 mM probenecid with 4.33 microM scutellarin, the Papp is 2.24 times than that of the control rats (p < 0.01). As shown by in silico experiments the spatial structure of scutellarin was in good agreement with the pharmacophore of Mrp2. The efflux of Mrp2, not P-gp, in the intestinal of the rats may be one of the reasons that lead to the low oral bioavailability of scutellarin.