The major drawbacks of P-glycoprotein (P-gp) inhibitors at the clinical stage make the development of new P-gp inhibitors challenging and desirable. In this study, we reported our structure-activity relationship studies of 4-indolyl quinazoline, which led to the discovery of a highly effective and orally active P-gp inhibitor, <b>YS-370</b>. <b>YS-370</b> effectively reversed multidrug resistance (MDR) to paclitaxel and colchicine in SW620/AD300 and HEK293T-ABCB1 cells. <b>YS-370</b> bound directly to P-gp, did not alter expression or subcellular localization of P-gp in SW620/AD300 cells, but increased the intracellular accumulation of paclitaxel. Furthermore, <b>YS-370</b> stimulated the P-gp ATPase activity and had moderate inhibition against CYP3A4. Significantly, oral administration of <b>YS-370</b> in combination with paclitaxel achieved much stronger antitumor activity in a xenograft model bearing SW620/Ad300 cells than either drug alone. Taken together, our data demonstrate that <b>YS-370</b> is a promising P-gp inhibitor capable of overcoming MDR and represents a unique scaffold for the development of new P-gp inhibitors.