We examined the expression of the transcription factor NF‐κB, a nuclear trans‐acting factor known to play a key role in cytokine gene regulation, in patients with inflammatory bowel disease (IBD). It was found that LP macrophages in Crohn's disease (CD) and ulcerative colitis (UC) display high levels of NF‐κB DNA‐binding activity accompanied by an increased production of interleukin (IL)‐1, IL‐6, and tumor necrosis factor (TNF)α. Western blot studies showed an increased expression of the p50 and c‐rel subunits of NF‐κB; however, the most striking finding was an increased expression level of NF‐κB p65 in patients with CD and UC. Selective downregulation of p65 in IBD macrophages by a specific antisense phosphorothioate oligonucleotide was sufficient to considerably reduce production of proinflammatory cytokines. These results demonstrate a characteristic increase of NF‐κB binding levels in patients with IBD. The data suggest that antisense DNA targeting NF‐κB p65 can be used as a novel molecular approach for the treatment of patients with IBD.