Evidence is accumulating that, in addition to regulating peripheral energy metabolism, insulin is an important modulator of neuronal function. Indeed, high levels of insulin and insulin receptors are expressed in several brain regions including the hippocampus. We have shown previously that insulin inhibits aberrant synaptic activity in hippocampal neurons via activation of large conductance Ca2+-activated K+ (BK) channels. In this study, we have examined further the effects of insulin on native hippocampal and recombinant (hS/o) BK channels expressed in human embryonic kidney (HEK) 293 cells. Pipette or bath application of insulin evoked a rapid increase in hippocampal BK channel activity, an action caused by activation of insulin receptors because insulin-like growth factor 1 (IGF-1) failed to mimic insulin action. In parallel studies, insulin, applied via the pipette or bath, also activated hS/o channels expressed in HEK293 cells. Although phosphoinositide 3-kinase is a key component of insulin and IGF-1 receptor signaling pathways, activation of this lipid kinase does not underlie the effects of insulin because neither 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) nor wortmannin inhibited or reversed insulin action. However, specific inhibitors of mitogen-activated protein kinase (MAPK) activation, 2′-amino-3′ -methoxyflavone (PD98059) or 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126), attenuated insulin action, indicating that a MAPK-dependent mechanism underlies this process. Furthermore, insulin activation of this pathway enhances BK channel activity by shifting the Ca2+-sensitivity such that BK channels are active at more hyperpolarized membrane potentials. Because postsynaptic BK channels are important regulators of neuronal hyperexcitability, insulin-induced activation of BK channels, via stimulation of a MAPK-dependent pathway, may be an important process for regulating hippocampal function under normal and pathological conditions.