Naegleria fowleri is pathogenic free-living amoeba leading to primary amoebic meningoencephalitis(PAM) in human and animals. The nfa1 gene cloned from N. fowleri is located on food-cup structure called pseudopodia and function an adherence of host target cells. To evaluate the effect of nfa1 vaccination against N. fowleri infection, we constructed retroviral vector(pQCXIN) expressing nfa1 gene. To determine the effect of vaccination and protective immunity in in vivo models, we measured the immunoglobulin levels, cytokine induction, and survival rate in mice infected with N. fowleri. Both levels of IgG and IgG subclass in DNA vaccinated mice were significantly elevated. The cytokine analysis show that DNA vaccinated mice induces production of IL-4 and IFN-γcytokines suggesting a Th1/Th2 mixed type immune response. The levels of nfa1 specific IgG antibody were maintained highly until 12 weeks post-vaccination in vaccinted mice. The nfa1 vaccinated mice using retroviral vector increased significantly survival rate(60%) after N. fowleri infection. Consequently, the nfa1 vaccination effectively induces protective immunity by upregulation of immune response in mice infected with N. fowleri. These results suggest that DNA vaccination using retroviral vector may be proper trial for treatment and prevention of PAM.