We have found that giant hornets (Vespa mandarinia japonica) are killed in less than 10 min when they are trapped in a bee ball created by the Japanese honeybees Apis cerana japonica, but their death cannot be solely accounted for by the elevated temperature in the bee ball. In controlled experiments, hornets can survive for 10 min at the temperature up to 47°C, whereas the temperature inside the bee balls does not rise higher than 45.9°C. We have found here that the CO 2 concentration inside the bee ball also reaches a maximum (3.6 ± 0.2%) in the initial 0–5 min phase after bee ball formation. The lethal temperature of the hornet (45–46°C) under conditions of CO 2 concentration (3.7 ± 0.44%) produced using human expiratory air is almost the same as that in the bee ball. The lethal temperature of the honeybee is 50–51°C under the same air conditions. We concluded that CO 2 produced inside the bee ball by honeybees is a major factor together with the temperature involved in defense against giant hornets.