AbstractEntosis is a non‐apoptotic cell death process that forms characteristic cell‐in‐cell structures in cancers, killing invading cells. Intracellular Ca2+dynamics are essential for cellular processes, including actomyosin contractility, migration, and autophagy. However, the significance of Ca2+and Ca2+channels participating in entosis is unclear. Here, it is shown that intracellular Ca2+signaling regulates entosis via SEPTIN‐Orai1‐Ca2+/CaM‐MLCK‐actomyosin axis. Intracellular Ca2+oscillations in entotic cells show spatiotemporal variations during engulfment, mediated by Orai1 Ca2+channels in plasma membranes. SEPTIN controlled polarized distribution of Orai1 for local MLCK activation, resulting in MLC phosphorylation and actomyosin contraction, leads to internalization of invasive cells. Ca2+chelators and SEPTIN, Orai1, and MLCK inhibitors suppress entosis. This study identifies potential targets for treating entosis‐associated tumors, showing that Orai1 is an entotic Ca2+channel that provides essential Ca2+signaling and sheds light on the molecular mechanism underlying entosis that involves SEPTIN filaments, Orai1, and MLCK.