The nuclear factor-kappa B (NF-κB) family of transcription factors regulate gene expression in response to diverse stimuli. We previously demonstrated that valproic acid (VPA) exposure in utero decreases total cellular protein expression of the NF-κB subunit p65 in CD-1 mouse embryos with a neural tube defect but not in phenotypically normal littermates. This study evaluated p65 mRNA and protein expression in P19 cells and determined the impact on DNA binding ability and activity. Exposure to 5mM VPA decreased p65 mRNA and total cellular protein expression however, nuclear p65 protein expression was unchanged. VPA reduced NF-κB DNA binding and nuclear protein of the p65 DNA-binding partner, p50. NF-κB transcriptional activity was increased with VPA alone, despite decreased phosphorylation of p65 at Ser276, and when combined with tissue necrosis factor α. These results demonstrate that VPA increases NF-κB transcriptional activity despite decreasing DNA binding, which may play a role in VPA-initiated teratogenesis.