The effectiveness of cancer chemotherapy is often circumvented by multidrug 
resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug 
transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor 
receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously 
capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in 
human cancer cells. Furthermore, some TKIs are transported by ABCB1, which 
results in low oral bioavailability, reduced distribution, and the development 
of acquired resistance to these TKIs. In this study, we investigated the 
interaction between ABCB1 and osimertinib, a novel selective, irreversible 
third-generation EGFR TKI that has recently been approved by the U.S. Food and 
Drug Administration. We also evaluated the potential impact of ABCB1 on the 
efficacy of osimertinib in cancer cells, which can present a therapeutic 
challenge to clinicians in the future. We revealed that although osimertinib 
stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer 
resistance to osimertinib. Our results suggest that it is unlikely that the 
overexpression of ABCB1 can be a major contributor to the development of 
osimertinib resistance in cancer patients. More significantly, we revealed an 
additional action of osimertinib that directly inhibits the function of ABCB1 
without affecting the expression level of ABCB1, enhances drug-induced 
apoptosis, and reverses the MDR phenotype in ABCB1-overexpressing cancer cells. 
Considering that osimertinib is a clinically approved third-generation EGFR TKI, 
our findings suggest that a combination therapy with osimertinib and 
conventional anticancer drugs may be beneficial to patients with MDR tumors.