Naegleria fowleri, a pathogenic free-living amoeba has been isolated from samples obtatined from soil,polluted water and chlorinated swimming pool waster and existed as a virulent pathogen which causes fatal primary amoebic meningoencephalitis (PAM) in experimental animal or humans. The cytotoxicity of N. fowleri on target cells requires the contact-dependent mechanism such as a phagocytosis and the contact-independent mechanism such as a secretion of proteases. The phagocytosis mechanisms involve a process of piecemeal ingestion of target cells by food-cups (amoebastomes). Phagocytosis is an actin dependent process that includes polymerization of monomeric G-actin into filamentous F-actin. Despite the numerous studies concerning with phagocytosis, the detailed role of actin in the N. fowleri food-cup formation has been poorly reported. In this study, we cloned and characterized an nf-actin gene for elucidation of the role of nf-actin in N. fowleri pathogenesis. The nf-actin gene is composed of 1,124 bp and the sequence identity was 82% with nonpathogenic N. gruberi. No sequence identity with other mammals or human actin gene. Immunofluorescence assay using anti-Nf-actin polyclonal antibody developed in BALB/c mice immunized with recombinant protein (rNf-actin fused with His-tag) revealed that the Nf-actin was localized in the cytoplasm and pseudopodia, especially, food-cup structure (amoebastome), of N. fowleri trophozoites. When N. fowleri were co-cultured with CHO cells, the Nf-actin was observed to localize around on phagocytic food-cups. When the Nf-actin protein was inhibited with cytochalasin D, the actin polymerization inhibitor, or nf-actin gene knock-downed by transfection with antisense oligomers, N. fowleri trophozoites showed reduced food-cup structures and in vitro cytotoxicity. It suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri. In addition, the nf-actin gene was inserted into Ubi-pEGFP-C2 vector for overexpression, and then Ubi-pEGFP-C2/nf-actin was transfected to N. fowleri trophozoites. The strong GFP fluorescence was observed in N. fowleri trophozoites transfected with Ubi-pEGFP-C2/nf actin, and the expression of EGFP-Nf-actin protein was detected by western blot. The nf-actin overexpressed transgenic N. fowleri showed significantly increasing adherence ability against extracellular matrix components such as fibronectin, collagen I and fibrinogen in comparison to wild type N. fowleri. Moreover, the nf actin overexpressed transgenic N. fowleri showed increasing phagocytotic ability and cytotoxicity in comparison with the wild type N. fowleri. Finally, these results suggest that the nf-actin gene plays an important role in cell adhesion, phagocytosis and cytotoxicity of N. fowleri.