Low intracellular K⁺ concentration ([K⁺]_i) promotes apoptosis and blocking K⁺ loss prevents apoptosis, but the mechanism of action of low [K⁺]_i remains unclear. Here, we show that low [K⁺]_i increases NF-κB transcriptional activity by enhancing its binding to the promoter of target genes without affecting its activation and nuclear translocation in cortical neurons deprived of serum. Low K⁺ concentration promotes NF-κB/DNA binding through direct effects on the interaction of NF-κB dimers with DNA. Up-regulation of proapoptotic protein Bcl-X_S and neuronal apoptosis induced by serum deprivation are blocked by inhibition and/or down-regulation of NF-κB and by prevention of K⁺ loss. Thus, a direct action of K⁺ on NF-κB/DNA binding regulates gene transcription related to neuronal apoptosis.