In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) is not known yet. The mouse B cells express large conductance background K+ channels (LKbg) that are inhibited by PIP2. In inside-out patch clamp studies, the application of MgATP (1 mm) also inhibited LKbg due to the generation of PIP2 by phosphoinositide (PI)-kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LKbg, which was antagonized by PIP2 (> 1 μm) or higher concentration of MgATP (5 mm). The inhibition by PIP2 was partially reversible. However, the application of methyl-β-cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LKbg activity and facilitated the activation by stretch. In cell-attached patches, LKbg were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano-activation of LKbg was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m-3M3FBS or by cross-linking IgM-type B cell receptors activated LKbg. Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI-231 induces tyrosine phosphorylation of PLCγ2 and PIP2 hydrolysis of plasma membrane. The time dependence of PIP2 hydrolysis and LKbg activation were similar. The presence of LKbg and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch-dependent ion channel activation, namely, that the degradation of PIP2 caused by stretch-activated PLC releases LKbg from the tonic inhibition by PIP2.