Fire ants are widely studied, invasive and venomous arthropod pests. There is significant biomedical interest in immunotherapy against fire ant stings. However, mainly due to practical reasons, the physiological effects of envenomation has remained poorly characterized. The present study takes advantage of a recently-described venom protein extract to delineate the immunological pathways underlying the allergic reaction to fire ant venom toxins. Mice were injected with controlled doses of venom protein extract. Following sensitization and a second exposure, a marked footpad swelling was observed. Based on eosinophil recruitment and production of Th2 cytokines, we hereby establish that fire ant proteins per se can lead to an allergic response, which casts a new light into the mechanism of action of these toxins.