PAME caused by N. fowleri is an acute, fulminant, and rapidly progressing fatal illness that usually affects children and young adults. There have been few reports to address proteins functioning in vitro cytotoxicity of N. fowleri. The nfa1 gene, cloned from a cDNA library of N. fowleri by immunoscreening, should be concerned with the formation of food-cups that is a phagocytic structure. In addition, an anti-Nfa1 antobody reduced the in vitro cytotoxicity of N. fowleri against target cells. To elucidate the function of proteins cloned from N. fowleri, the gene-knockdown analysis by a transfection system are not yet established. In this present study, to describe the association of an Nfa1 protein in vitro cytotoxicity of N. fowleri to target cells, an antisense RNA or siRNA of nfa1 gene were transfected into N. fowleri trophozoites. By the synthetic dsRNA of nfa1 gene ORF, the expression of nfa1 gene and the Nfa1 protein were knockdowned about 50% and 30%, respectively. However, by antisense RNA transcribed in vitro, the expression of nfa1 gene and the Nfa1 protein were less knockdowned than those of dsRNA of nfa1 gene. Four synthetic siRNAs were not act equally, but a sinfa1-1 was highly effective to knockdown the nfa1 gene and Nfa1 protein with 70% and 43%, respectively. However, N. fowleri trophozoites transfected with synthetic dsRNA or sinfa1-1 did not highly induce in vitro cytotoxicity against murine macrophages as compared with normal N. fowleri trophozoites. Therefore, a vector-based system, in which transfected genes can be maintain longer, was used to transfect the nfa1 gene into N. fowleri. A pAct/SAGAH vector with a sinfa1-1 and a pAct/asnfa1AGAH vector with an asRNA of the nfa1 gene ORF were cloned, and then transfected into N. fowleri. By the pAct/SAGAH vector, the expression of nfa1 gene and the Nfa1 protein were knockdowned as 60% and 29%, in comparison with the pAct/asnfa1AGAH vector of 30% in the nfa1 gene and 18% in Nfa1 protein. In particular, the in vitro cytotoxicity of N. fowleri transfected with a pAct/SAGAH vector against macrophages was decreased to 26.6% at 17 h and 26.8% at 24 h post co-incubation, whereas the in vitro cytotoxicity was decreased to 7.4% at 17 h and 6.6% at 24 h by a pAct/asnfa1AGAH vector. These results suggest that the function of RNAi should be worked in N. fowleri trophozoites. Therefore, single stranded RNA, dsRNA, siRNA, and siRNA-vector were not only efficiently transfected into N. fowleri using each transfection reagent, but also decreased the function of Nfa1 protein which plays very important role in destroying macrophages. This result may be helpful for understanding the function of Nfa1 protein as a target cell-cantact mechanism in the N. fowleri infection.