Previous studies demonstrated that ING4 as a novel member of ING (inhibitor of growth) family has potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in inhibition of human tumors has not been reported. To explore its therapeutic effect on human lung carcinoma, we constructed a recombinant adenoviral vector Ad-ING4 expressing the humanized ING4 gene derived from murine ING4 with two amino acid modifications at residue 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of A549 human lung carcinoma cells induced cell apoptosis, altered cell cycle with S phase reduction and G2/M phase arrest, suppressed cell invasiveness, and down-regulated IL-6, IL-8, MMP-2, and MMP-9 expression of transfected tumor cells. In athymic mice bearing A549 lung tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth and reduced the tumor microvessel formation. Therefore, Ad-ING4 may be useful in gene therapy of human lung carcinoma.