AbstractMacropinocytosis (MPC) is a large‐scale endocytosis pathway that involves actin‐dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid‐phase cargos. MPC is categorized into two types: constitutive and stimuli‐induced. Constitutive MPC in macrophages relies on extracellular Ca²⁺ sensing by a calcium‐sensing receptor. However, the link between stimuli‐induced MPC and Ca²⁺ remains unclear. Here, we find that both intracellular and extracellular Ca²⁺ are required for epidermal growth factor (EGF)‐induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca²⁺ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca²⁺‐related regulator of EGF‐induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca²⁺, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C‐terminus known as a PDZ domain‐binding motif. Additionally, we show that ATP2B4‐KO reduces EGF‐stimulated Ca²⁺ oscillation during MPC. Our findings suggest that EGF‐induced MPC requires ATP2B4‐dependent Ca²⁺ dynamics.