KV2.1 is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium (KV) channel in mammalian central neurons, and is a target for activity-dependent modulation via calcineurin-dependent dephosphorylation. Using hanatoxin-mediated block of KV2.1 we show that, in cultured rat hippocampal neurons, glutamate stimulation leads to significant hyperpolarizing shifts in the voltage-dependent activation and inactivation gating properties of the KV2.1-component of delayed rectifier K+ (IK) currents. In computer models of hippocampal neurons, these glutamate- stimulated shifts in the gating of the KV2.1-component of IK lead to a dramatic suppression of action potential firing frequency. Current-clamp experiments in cultured rat hippocampal neurons showed glutamate stimulation induced a similar suppression of neuronal firing frequency. Membrane depolarization also resulted in similar hyperpolarizing shifts in the voltage-dependent gating properties of neuronal IK currents, and suppression of neuronal firing. The glutamate-induced effects on neuronal firing were eliminated by hanatoxin, but not by dendrotoxin-K, a blocker of KV1.1-containing channels. These studies together demonstrate a specific contribution of modulation of KV2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.