We assessed the role of the actin‐polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes. An in frame ΔactA mutant was constructed in a hyperinvasive strain of prfA* genotype, in which all genes of the PrfA‐dependent virulence regulon, including actA, are highly expressed in vitro. Loss of ActA production in prfA* bacteria reduced entry into Caco‐2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the ΔactA prfA* mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS‐1 fibroblasts and Hepa 1‐6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non‐invasive species into epithelial cell lines, but not into COS‐1 and Hepa 1‐6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco‐2 human enterocytes suggested that this pathway involves microvilli. prfA* bacteria, but not wild‐type bacteria (which express PrfA‐dependent genes very weakly in vitro) or prfA*ΔactA bacteria, efficiently invaded differentiated Caco‐2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA* strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin‐based motility and host cell tropism and invasion.