Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary their K⁺ channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV‐LPS) to determine the role of the voltage‐gated Kv1.3 channel in LPS‐induced M1‐like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV‐LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS‐induced microglial activation exemplified by Iba‐1 immunoreactivity and expression of pro‐inflammatory mediators such as IL‐1β, TNF‐α, IL‐6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS‐induced impairment of hippocampal long‐term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro‐inflammatory microglial neurotoxicity. Pharmacological intervention using PAP‐1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti‐inflammatory and hLTP‐recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1‐like pro‐inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia‐mediated neurotoxicity is implicated in the pathogenesis.