Deregulated activation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently found in human cancers, which plays a key role in promoting cancer proliferation and resistance to anticancer therapies. Therefore, developing inhibitors targeting key components of the PI3K/Akt/mTOR signaling pathway has great clinical significance. PF-4989216 is a novel, orally available small-molecule drug that was developed to selectively inhibit the PI3K/Akt/mTOR signaling pathway and subsequent cancer cell proliferation. PF-4989216 exhibited potent and selective inhibition against PI3K kinase activity in preclinical small-cell lung cancer (SCLC) models, and was especially effective against the proliferation of SCLCs harboring PIK3CA mutation. Unfortunately, in addition to innate resistance mechanisms, drug extrusion by the efflux pumps may also contribute to the development of acquired resistance to PI3K inhibitors in cancer cells. The overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and ABCG2 is one of the most common mechanisms for reducing intracellular drug concentration and developing multidrug resistance, which remains a substantial challenge to the effective treatment of cancer. In this study, we report the discovery of ABCG2 overexpression as a mechanism of resistance to PI3K inhibitor PF-4989216 in human cancer cells. We demonstrated that the inhibition of Akt and downstream S6RP phosphorylation by PF-4989216 were significantly reduced in ABCG2-overexpressing human cancer cells. Moreover, overexpression of ABCG2 in various cancer cell lines confers significant resistance to PF-4989216, which can be reversed by an inhibitor or competitive substrate of ABCG2, indicating that ABCG2-mediated transport alone can sufficiently reduce the intracellular concentration of PF-4989216.