Pathogenic Naegleria fowleri causes a fatal disease, called as primary amoebic menigoencephalitis, in humans and experimental animals. In the present study, to examine the protective immunity of the Nfa1 protein for N. fowleri infection in a mouse model, BALB/c mice were immunized with the Nfa1 protein either by intraperitoneal or intranasal route, and then infected intranasally with N. fowleri trophozoites. In regardless of the immunization routes, Nfa1-immunized mice showed the prolonged mean time to death. The specific immunoglobulin G (IgG), IgG subclass, IgA and IgE of mice induced against Nfa1 protein immunization was measured by ELISA. The levels of IgG and IgA were significantly increased in sera obtained from mice immunized with rNfa1 protein. Analysis of IgG subclass profiles revealed that IgG1 showed the greatest increasing followed by IgG2b, IgG2a and IgG3. In contrast, IgE level displayed low level of IgE similar to non-immunized mice. In addition, splenocytes of mice immunized with Nfa1 protein secreted significantly high levels of both gamma-interferon and interleukin-10 after stimulation with rNfa1 protein. These findings suggest that the rNfa1 protein may induce the humoral and cell-mediated immunity leading to the host defense in N. fowleri infection.