SummaryWe previously reported that intranasal administration of Cry1Ac protoxin alone or in combination with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice. Those results suggested that both antibody responses and innate immune mechanisms may be participating in the protective effects observed. The present study was aimed to investigate whether the STAT6‐induced Th2 immune response is essential for the resistance to N. fowleri infection, conferred by immunization with amoebic lysates plus Cry1Ac. STAT6‐deficient (STAT6−/−) and wild‐type (STAT6+/+) BALB/c mice were immunized by the intranasal route with a combination of N. fowleri lysates plus Cry1Ac, and subsequently challenged with lethal doses of N. fowleri trophozoites. STAT6+/+ mice displayed 100% protection, while no protection was observed in STAT6−/− mice. Significantly higher titres of Th2‐associated IgG1 as well as interleukin‐4 (IL‐4) were found in STAT6+/+ mice, whereas in STAT6−/− mice significantly more IL‐12 and IFN‐γ as well as significantly higher titres of Th1‐associated IgG2a were detected. Thus, whereas protected STAT6+/+‐immunized mice elicited a Th‐2 type inclined immune response that produced predominantly humoral immunity, unprotected STAT6−/− mice exhibited a polarized Th1 type cellular response. These findings suggest that the STAT6‐signalling pathway is critical for defence against N. fowleri infection.