The chemokine stromal cell-derived factor-1α (SDF-1α) has multiple effects on neuronal activity, survival, and death under conditions that generate a proinflammatory microenvironment within the brain, via signaling through C-X-C-type chemokine receptor 4 (CXCR4), although the underlying cellular/molecular mechanisms are unclear. Using rat hippocampal neurons, we investigated distinct modifications in the voltage-gated K⁺ (Kv) channel Kv2.1 in response to short- and long-term SDF-1α/CXCR4-mediated signaling as an underlying mechanism for CXCR4-dependent regulation of neuronal survival and death. Acute exposure of neurons to SDF-1α led to dynamic dephosphorylation and altered localization of Kv2.1 channel, resulting in enhanced voltage-dependent activation of Kv2.1-based delayed-rectifier Kv currents (I(DR)). These changes were dependent on CXCR4- and/or NMDA receptor-mediated activation of calcineurin and provide neuroprotection. However, prolonged SDF-1α treatment leads to CXCR4-mediated activation of p38 mitogen-activated protein kinase, resulting in phosphorylation of Kv2.1 at S800 and enhanced surface trafficking of the channel protein, resulting in increased I(DR)/Kv2.1 current density. This, in combination with sustained dephosphorylation-induced enhancement of the voltage-dependent activation of I(DR)/Kv2.1, predisposed neurons to excessive K⁺ efflux, a vital step for the neuronal apoptotic program. Such apoptotic death was dependent on CXCR4 and Kv2.1 function and was absent in cells expressing the Kv2.1-S800A mutant channel. Furthermore, similar modifications in Kv2.1 and CXCR4/Kv2.1-dependent apoptosis were observed following treatment of neurons with the human immunodeficiency virus-1 (HIV-1) glycoprotein gp120. Therefore, distinct modifications in Kv2.1 in response to short- and long-term CXCR4-mediated signaling could provide a basis for neuroprotection or apoptosis in neuropathologies, such as neuroinflammation, stroke, brain tumors, and HIV-associated neurodegeneration.