The apple snail Pomacea canaliculata is a freshwater snail originating in tropical and temperate South America. We investigated the effect of temperature acclimatization, moisture levels, and food availability on the loss of cold hardiness in overwintering snails. Cold hardiness broke down by four days in snails maintained in aquatic condition at 25°C and by eight days at 20°C. However, snails held at 15°C retained their cold hardiness even after 64 days. In addition, the cold hardiness of snails kept moist (wrapped in wet towel) was maintained for at least 64 days even at 25°C. These results indicate that warm temperature together with increase of water availability decreases the cold hardiness of overwintering snails. Glycerol content reduced as cold hardiness decreased; supporting a previous hypothesis that glycerol has a major physiological role in the development of cold hardiness in this species.