P-glycoprotein (P-gp), which is encoded by the ATP-binding cassette (ABC) transporter subfamily B member 1 (ABCB1) gene, is one of the most pivotal ABC transporters that transport its substrates across the cell membrane. Its overexpression is one of the confirmed causes of multidrug resistance (MDR), which results in the failure of cancer treatment. Here, we report that checkpoint kinase (Chk) 1 inhibitor MK-8776, a drug candidate in clinical trial, can restore the sensitivity of chemotherapeutics that are substrates of P-gp in KB-C2, SW620/Ad300 cells and human embryonic kidney (HEK)293/ABCB1 cells that overexpress P-gp. MK-8776 remarkably enhanced the cellular [³H]-paclitaxel accumulation and suppressed the efflux function of P-gp without reducing its expression and affecting its cellular localization in cancer cells. Furthermore, MK-8776 (0–40 μM) stimulated the activity of ATPase in P-gp, which was 4.1-fold greater than the control. In addition, MK-8776 formed a cation–π bond and π–π interaction with key residues of the substrate-binding site in P-gp, as indicated by computer-aided molecular docking study. Our study indicated that MK-8776 may significantly enhance the sensitivity of chemotherapeutics that are substrates of P-gp, providing important information for its application in the reversal of MDR.