A recent study in our laboratory has shown that an increase in intrathoracic temperature activates vagal pulmonary C-fibers. Because these afferents are known to elicit reflex bronchoconstriction upon stimulation, this study was carried out to investigate if increasing airway temperature within the physiological range alters the bronchomotor tone. Adult guinea pigs were anesthetized and mechanically ventilated via a tracheal tube. After the lung was hyperventilated with humidified hot air (HHA) for 4 min, the tracheal temperature was elevated from 36.4 to 40.5°C, which induced an immediate bronchoconstriction, increasing total pulmonary resistance (R_L) to 177 ± 10% and decreasing dynamic lung compliance to 81 ± 6% of their respective baselines. The increase in R_L returned spontaneously toward the baseline in <10 min, and was reproducible in the same animals. There was no difference in the response whether the humidity was generated from distilled water or isotonic saline. In contrast, hyperventilation with humidified air at room temperature did not cause any increase in R_L. The increase in R_L caused by HHA (R_L) was attenuated by 65.9% after a pretreatment with atropine alone, and by 72.0% after a pretreatment with a combination of atropine and neurokinin receptor type-1 and -2 antagonists. In addition, capsazepine, a selective transient receptor potential vanilloid type 1 (TRPV1) antagonist, reduced the HHA-induced increase in R_L by 64.1%, but did not abolish it. However, a pretreatment with formoterol, a ₂ agonist, completely prevented the HHA-induced bronchoconstriction. These results indicate that increasing airway temperature induced a transient airway constriction in guinea pigs. Approximately two-third of the increase in bronchomotor tone was mediated through the cholinergic reflex that was probably elicited by activation of TRPV1-expressing airway afferents. The remaining bronchoconstriction was caused by other yet unidentified factors.