Phosphoinositide 3-kinase (PI3-kinase) has been shown to link leptin receptor activation to stimulation of large conductance Ca2+-activated K+ (BK) channels and subsequent inhibition of hippocampal epileptiform-like activity. However, the downstream targets of PI3-kinase in this action of leptin are unknown. Here we show that BK channel activation by leptin is dependent on the actin cytoskeleton, as it is prevented by actin filament stabilization and mimicked by actin disruption. Fluorescent labeling of polymerized actin filaments revealed that leptin promotes the rapid rearrangement of actin filaments via activation of PI 3-kinase; an action paralleled by discrete increases in PtdIns(3,4,5)P3 immunoreactivity in close proximity to BK channels. After leptin exposure, there was also an actin-dependent increase in the association of BK channel immunoreactivity with synaptic markers. These data are consistent with the notion that leptin activates BK channels via PI 3-kinase-dependent reorganization of actin filaments and subsequent clustering of BK channels at synapses.