Phosphatidylinositol 3-kinase α is an attractive target to potentially treat a range of cancers. Herein, we described the evolution of a reported PI3K inhibitor into a moderate PI3Kα inhibitor with a low molecular weight. We used X-ray crystallography to describe the accurate binding mode of the compound YXY-4F. A comparison of the p110α–YXY-4F and apo p110α complexes showed that YXY-4F induced additional space by promoting a fexible conformational change in residues Ser773 and Ser774 in the PI3Kα ATP catalytic site. Specifcally, residue 773(S) in PI3Kα is quite diferent from that of PI3Kβ (D), γ (A), and δ (D), which might guide further optimization of substituents around the NH group and phenyl group to improve the selectivity and potency of PI3Kα.