Class IB phosphoinositide 3-kinase gamma (PI3Kγ) is vital for regulating intracellular signaling pathways and has become an attractive drug target for the treatment of malignant tumors. In the present study, one potent PI3Kγ inhibitor (JN-PK1) with a novel scaffold against hematologic malignancies was identified based on a series of biological experiments, and then the selective mechanism of PI3Kγ inhibition was explored by a systematic computational method. JN-PK1 shows an effective antiproliferative activity on several cancer cell lines, especially blood cancer cells. Cell-free enzymatic studies demonstrated that JN-PK1 specifically inhibits PI3Kγ at low micromolar concentrations without affecting other isoforms of PI3K. In the cellular context, JN-PK1 potently inhibits PI3K/Akt/mTOR signaling pathway in a time- and concentration-dependent manner, which leads to the apoptosis of cancer cells. Further, the specific binding mode of JN-PK1 with PI3Kγ was illustrated by molecular docking, and the selective inhibition mechanism of PI3Kγ by JN-PK1 was revealed by molecular dynamics simulation. Finally, some key residues of PI3Kγ required for specificity and activity were identified. Taken together, JN-PK1 may be developed as a promising therapeutic agent for the treatment of hematologic malignancies.