Microsporidia are spore-forming obligate intracellular eucaryotes that parasitize eukaryotic cells. Encephalitozoon intestinalis (formerly Septata intestinalis) is a microsporidian species of emerging medical importance, responsible for chronic diarrhoea in immunocompetent patients and enteritis and systemic infections in HIV-1 infected patients. Infection of host enterocytes has been demonstrated in HIV-1-infected patients. However, the mechanisms of entry of E. intestinalis into host enterocytes have not been studied and remain hypothetically based on diacytosis, a model involving the injection of microsporidian sporoplasm through the polar tubule into the host cell. An electron microscopy based study recently challenged this hypothesis. We studied the entry of E. intestinalis into intestinal epithelial cells by infecting the human enterocyte-like cell line Caco-2. Entry was mediated by directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on E. intestinalis uptake, colocalization of E. intestinalis and F-actin and engulfment of E. intestinalis into Caco-2 cell protrusions. Confocal- and electron microscopy observations also suggested that after initial contacts through the posterior pole of the microsporidian spore, the basolateral surface of Caco-2 cells may be the portal of entry for E. intestinalis sporoplasm. Our observations allowed us to propose a new, actin-based model to describe the entry of microsporidia into enterocytes.