The Hippo pathway plays a key role in development, organ size control and tissue homeostasis, and its dysregulation contributes to cancer. The LATS tumor suppressor kinases phosphorylate and inhibit the YAP/TAZ transcriptional co-activators to suppress gene expression and cell growth. Through a screen of marine natural products, we identified microcolin B (MCB) as a Hippo activator that preferentially kills YAP-dependent cancer cells. Structure–activity optimization yielded more potent MCB analogs, which led to the identification of phosphatidylinositol transfer proteins α and β (PITPα/β) as the direct molecular targets. We established a critical role of PITPα/β in regulating LATS and YAP. Moreover, we showed that PITPα/β influence the Hippo pathway via plasma membrane phosphatidylinositol-4-phosphate. This study uncovers a previously unrecognized role of PITPα/β in Hippo pathway regulation and as potential cancer therapeutic targets.