Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin-dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P2 as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G-actin unlike PIPKs from other organisms. PtdIns(4,5)P2, the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP-tagged PH-domain from PLCδ, which specifically binds PtdIns(4,5)P2 in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics. © 2019 John Wiley & Sons Ltd