Intracellular Ca²⁺ signaling plays obligatory roles in the regulation of cellular physiological functions, and also the proliferation and/or death of cells. Hyper-activation of Ca²⁺ signaling often initiates and/or facilitates the pathological conditions in many kinds of diseases and has been studied as the potential target for new therapeutic approaches and drug discovery. The generation of Ca²⁺ signaling has two major pathways; the Ca²⁺ inflow and the Ca²⁺ release from intracellular stores, which are controlled in extensively different manners depending on cellular excitability. In excitable cells, membrane potential changes due to ion channel activities and Ca²⁺ signaling are bidirectionally regulated to maintain the homeostasis of physiological functions efficiently by Ca²⁺ microdomain formation, which has been revealed by molecular imaging. Even in non-excitable cells, the resting membrane potential substantially modulates Ca²⁺ signaling via the inflow through non-voltage-dependent Ca²⁺ channels, such as store-operated Ca²⁺ entry. The crucial significance of K⁺ channel in Ca²⁺ signaling is attributable to its inverse functions in excitable and non-excitable cells. Based on the large molecular diversity, the heterologous multimer-formation and the tissue-specific expression pattern, K⁺ channels particularly in non-excitable cells are now recognized as a hot target of drug discovery research and development.