During insect development from embryo to adult, airway clearance in tracheal system occurs episodically each time the molt is completed by performance of the ecdysis sequence. We found that the neuropeptide Kinin is required for normal respiratory fluid clearance or “tracheal air-filling” in fruit fly Drosophila larvae. Disruption of Kinin signaling leads to defective air-filling during all larval stages. Targeted Kinin receptor silencing in tracheal epithelial cells also shows tracheal air-filling defect. On the other hand, promotion of Kinin signaling in vivo through peptide injection or Kinin neuron activation induces premature tracheal collapse and air-filling. Moreover, direct exposure of epithelial cells in vitro to Kinin leads to calcium mobilization in tracheal epithelial cells. Our findings strongly implicate the neuropeptide Kinin as an important regulator of airway clearance via intracellular calcium mobilization in tracheal epithelial cells of fruit fly Drosophila.