To explore mechanisms controlling sonic hedgehog (Shh) expression in human cancers, we investigated regulation of Shh by the transcription factor NF‐κB. We identify putative NF‐κB binding sites in the human Shh promoter region that specifically bind NF‐κB complexes. Further, NF‐κB activation by tumor necrosis factor α (TNF‐α) or p65 overexpression stimulates Shh promoter activity and p65 binds to Shh promoter in vivo. NF‐κB‐mediated transcriptional activation of Shh is mapped to a minimal NF‐κB consensus site at position +139 of Shh promoter. NF‐κB activation results in increased Shh mRNA and protein expression in vitro and, notably, also in vivo in a genetic mouse model of inducible NF‐κB activity. Specific NF‐κB inhibition by inhibitory NF‐κBα (Iκ‐Bα) superrepressor or p65 knockdown inhibits NF‐κB‐induced Shh promoter activation and Shh expression. NF‐κB‐mediated Shh expression promotes proliferation and confers resistance to TRAIL‐induced apoptosis. Silencing of Shh prevents NF‐κB‐stimulated proliferation, while the addition of Shh rescues the proliferation defect imposed by NF‐κB inhibition. Notably, NF‐κB‐stimulated tumor growth is significantly impaired by Shh knockdown in an in vivo model of pancreatic cancer. By demonstrating that NF‐κB regulates Shh expression, which contributes to NF‐κB‐mediated proliferation and apoptosis resistance in vitro and in vivo, our findings have important implications to target aberrant Shh expression in human cancers.—Kasperczyk, H., Baumann, B., Debatin, K.‐M., Fulda, S. Characterization of sonic hedgehog as a novel NF‐κB target gene that promotes NF‐κB‐mediated apoptosis resistance and tumor growth in vivo. FASEB J. 23, 21‐33 (2009)