Among the several types of cells composing the airway epithelium, the ciliary cells are responsible for one of the most important defense mechanisms of the airway epithelium: the transport of inhaled particles back up into the throat by coordinated ciliary movement. Changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) are the main driving force controlling the ciliary activity. In mouse ciliary cells, membrane hyperpolarization from −20 to −60 mV under whole-cell voltage-clamp induced a slow but significant [Ca²⁺]_i rise in a reversible manner. This rise was completely inhibited by the removal of Ca²⁺ from the extracellular solution. Application of diazoxide, an ATP-dependent K⁺ channel opener, dose-dependently induced a membrane hyperpolarization (EC₅₀ = 2.3 μM), which was prevented by the addition of 5 μM glibenclamide. An inwardly rectifying current was elicited by the application of 10 μM diazoxide and suppressed by subsequent addition of 5 μM glibenclamide. Moreover, the application of 10 μM diazoxide induced a significant [Ca²⁺]_i rise and facilitated ciliary movement. Multi-cell reverse-transcription polymerase chain reaction analyses and immunocytochemical staining suggested that the subunit combination of Kir6.2/SUR2B and possibly also Kir6.1/SUR2B is expressed in ciliary cells. The confocal Ca²⁺ imaging analyses suggested that the [Ca²⁺]_i rise induced by diazoxide occurred preferentially in the apical submembrane region. In conclusion, the application of a K_ATP channel opener to airway ciliary cells induces membrane hyperpolarization and thereby induces a [Ca²⁺]_i rise via the facilitation of Ca²⁺ influx through the non–voltage-dependent Ca²⁺ permeable channels. Therefore, a K_ATP opener may be beneficial in facilitating ciliary movement.