To minimize predation risk and the cost of confronting predators, prey have developed a range of defensive strategies and warning signals. Although advantageous, defensive warnings may also induce physiological and energy costs to the emitter. Ventilatory sounds (hissing) are the most distributed warning sound in vertebrates. Because they involve the respiratory apparatus, defensive hissing may substantially increase evaporative water loss. Herein, we examined the determinants of hissing as well as its physiological costs in a medium-sized venomous snake, the long-nosed viper (Vipera ammodytes). We first used a neutral arena and applied standardized stimulation to measure the occurrence and acoustic characteristics of warning hissing. Then, we used open-flow respirometry to quantify changes in respiratory gas exchanges (oxygen consumption and evaporative water loss) during defensive responses. We demonstrated that males are more likely to engage in sound warnings when stimulated. Expirations generated the strongest signals compared to inspiration but did not differ between sexes. We found that defensive hissing dramatically increased average metabolic rate and evaporative water loss during the 10-minute stimulation period, and this effect was more pronounced in males. Metabolic rates and evaporative water loss were closely related to the duration of hissing. Overall, our results indicate that respiratory-based warning sounds induce significant physiological costs and may alter water balance. The higher responsiveness in males than females likely reflects sexually selective pressure (higher mobility for mate acquisition) and enhanced risk exposure.