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Objective: To explore the mechanism of calcium-sensing receptor (CaSR) in hypoxia-induced airway mucous hypersecretion., Methods: Cultured human airway epithelial cells HBE16 by hypoxia incubator (94%N₂, 1%O₂, 5%CO₂, 37 °C). HBE16 were transfected with CaSR targeted small interfering RNA (CaSR-siRNA), pretreated with a specific activator of CaSR CaCl₂ and preincubated with various inhibitors [Gαq/11 protein inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, inositol 1, 4, 5-triphosphate receptors (IP 3R) inhibitor 2-APB and cell-permeable intracellular calcium chelator BAPTA-AM] before hypoxia. The level of MUC5AC mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). And the relative content of CaSR protein was detected by Western blot. The relative content of MUC5AC protein was measured by enzyme-linked immunosorbent assay (ELISA). The intracellular calcium concentration ([Ca²⁺]i) was measured by laser confocal microscopy., Results: The relative contents of [Ca²⁺]i and MUC5AC protein were obviously higher in hypoxic group ((154.2 ± 11.4) nmol/L, (0.624 ± 0.063) µg/L) than those in control group ((67.5 ± 2.8) nmol/L, (0.257 ± 0.051) µg/L) (all P < 0.01). The relative levels of CaSR protein and MUC5AC mRNA were significantly higher in hypoxic group (0.423 ± 0.028, 0.736 ± 0.045) than those in control group (0.185 ± 0.036, 0.321 ± 0.034) (all P < 0.01). CaCl₂ enhanced the effect of hypoxia. And the results had statistical significances (all P < 0.01). Transfection with CaSR-siRNA significantly decreased the effect of hypoxia (all P < 0.01). Pretreatments with Gαq/11 protein inhibitor, PLC inhibitor, IP 3R inhibitor or intracellular calcium chelator all significantly attenuated the hypoxia-induced MUC5AC hypersecretion and [Ca²⁺]i (all P < 0.05)., Conclusion: CaSR mediates hypoxia-induced airway mucous hypersecretion through a signaling pathway of Gαq/11/PLC/inositol 1, 4, 5-triphosphate (IP 3)/[Ca²⁺]i.