Phosphatidylinositol 3-kinase beta (PI3Kβ) is the dominant isoform of PI3K and has been implicated inthrombosis as well as phosphatase and tensin homologue-loss-induced tumorigenesis. PI3Kβhas beenconsidered to be an attractive target for anticancer drug discovery, and several PI3Kβinhibitors have pro-gressed into clinical trials. Here, we disclose the discovery of two natural products (PBY-0002 and PBY-0006) that have inhibitory effects on PI3Kβ. These two natural products were identified throughpharmacophore-based virtual screening, molecular docking, and a molecular dynamics simulation. Fur-thermore, anin vitroassay against human gastric cancer cell lines revealed that these two compoundsshowed anticancer activity. To identify the binding modes of PBY-0002 and PBY-0006 further, we per-formed a systematical investigation with comparison to the binding mode of GSK2636711, which is aknown PI3Kβinhibitor. The results demonstrated that PBY-0002 and PBY-0006 were tightly embeddedinto the ATP-binding site via hydrogen bonds andπ-cation interactions. These two natural products canprovide a promising starting point for the rational design of potent analogs with inhibitory activityagainst PI3Kβ.