Silkworm pupae are edible insects with high-quality nutrition in many Asian countries, but consumption of silkworm pupae can cause severe IgE-mediated allergic disease. The aim of this study was to investigate the effect of heat, enzymatic hydrolysis and acid-alkali treatment on the allergenicity of silkworm pupa protein extract (SPPE). Heating reduced the allergenicity of SPPE when the temperature was higher than 60 °C. Spectroscopy studies suggested an unfolded conformation of SPPE with heating, dependent on temperature and time. Enzymatic hydrolysis revealed that SPPE at 25 to 33 kDa contained pepsin- and trypsin-resistant allergens. The results of acid-alkali treatment suggested that low pH can promote hydrolysis of SPPE and decrease its allergenicity. Thus, heat, enzymatic hydrolysis and acid-alkali treatment can significantly decrease the allergenicity of SPPE, with heat-, enzyme- and acid-alkali-resistant allergens at 25 to 33 kDa SPPE. This study can help in the development of methods to prepare silkworm pupa protein.