Background Nuclear factor κB (NF-κB) overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant (IκBαM) gene, constructed and characterized its replication-deficient recombinant adenovirus (AdIκBαM), and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells. Methods IκBαM gene (203-1003 bp) encoding 267 amino acids, acquired by site-directed deleting N-terminal phosphorylation sites of serine 32/36, was subcloned into the pShuttle and pGEM-T vectors for further polymerase chain reaction (PCR), restriction digestion, deoxyribonucleic acid (DNA) sequencing and homology analyses. Subsequent to inserting the expression unit of pShuttle-IκBαM, containing cytomegalovirus (CMV) promoter, IκBαM complementary DNA (cDNA) and polyadenylic acid (PolyA) signals, into the type 5 adenovirus (Ad5) vector, the resultant AdIκBαM was packaged in human embryonic kidney (HEK) 293 cells by cotransfection with lipofectamine. Western blot analysis and electrophoretic mobility shift assay were utilized to detect the AdIκBαM-mediated overexpression of IκBαM in HEK293 cells and its suppressive effect on phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation in human umbilical vein endothelial (ECV304) cells, respectively. Results The relevant nucleotides and deduced amino acids of 801 bp IκBαM gene were consistent with those of IκBα gene (GenBank accession number: M69043). The titer of the prepared AdIκBαM was 4.0×1012 plaque-forming units (pfu)/L. Moreover, the IκBαM gene was overexpressed in HEK293 cells, and potently inhibited the PMA-induced NF-κB activation in ECV304 cells dose-dependently. Conclusions AdIκBαM is a novel vector for both efficient transfer and specific overexpression of IκBαM gene, as well as potent inhibition of NF-κB activity, providing a promising strategy for gene therapy of asthma.