Significance Macropinocytosis is a form of endocytosis that is accompanied by ruffling of plasma membrane and participates in a diverse range of pathophysiological processes, such as antigen uptake by immune cells and tumor growth. However, the molecular mechanism underlying this process is poorly understood. By exploiting the studies of fluid-phase endocytosis in Caenorhabditis elegans , we found that dephosphorylation of phosphoinositide PI(3)P is essential for macropinocytosis in mammalian cells. We also found that the sequential dephosphorylation of PI(3,4,5)P 3 → PI(3,4)P 2 → PI(3)P → PI at membrane ruffles is required for macropinocytosis. Identification of phosphoinositide phosphatases in the dephosphorylation cascade and a PI(3)P-sensitive K + channel as essential factors for macropinocytosis may provide the way to selectively control macropinocytosis among various endocytic pathways.