Objective The aim of this study was to explore the effects of spray cryotherapy (SCT) on cough receptors and airway microenvironment in a canine model of chronic bronchitis. Methods We examined the expression of transient receptor potential channels (TRPs) and neuropeptides at the gene and protein levels before and after SCT to see whether they further regulate the level of inflammatory mediators and cytokines via the membrane protein proteinase-activated receptor-2 / phospholipase-C?3 (PAR2/PLC3). In addition, we explored whether TRPs and neuropeptides co-localized in cultured rat dorsal root neurons after cigarette- smoking extract (CSE) stimulation by immunofluorescence; and whether TRPs could regulate inflammatory factors via mediator ATP. The levels of ATP and cytokines in alveolar lavage fluid and cell supernatant were measured using ELISA. Results SCT effectively down-regulated the expression of TRPs and neuropeptides in airway tissues of canines with chronic bronchitis, and further lowered the level of inflammatory mediators and cytokines that affect cough receptor sensitivity to achieve cough relief. Co-localization of TRPs and neuropeptide SP / CGRP was observed in CSE-stimulated rat dorsal root neurons. TRPS - ATP - inflammatory cytokines axis has been shown in cells, which in turn modulate the milieu of the airways and promote the formation of a cough feedback loop. Conclusions Our study fully revealed the specific mechanism of SCT, which involves successfully reducing the expression of cough receptors and neuropeptides distributed in the airway tissue and improving the airway inflammation microenvironment in order to ultimately achieve cough relief.