The expression of Ornithine decarboxylase (ODC) which is the first key enzyme of polyamine biosynthesis is increased in cancer cells. We had blocked the polyamine synthesis pathway using the adenoviral-mediated antisense ODC in some cancer cells such as prostate cancers and colorectal cancers. These researches demonstrated that ODC antisense expression could inhibit tumor cell growth. In order to reach the goal of applying the targeting gene therapy in clinical practice, we cloned the antisense ODC RNA which was driven by cancer specific promoter (hTERT promoter; telomerase reverse transcriptase promoter) into the adenovirus vector (rAd-CMV-GFP-hTERTp-ODC). Human cancer cell lines (HepG2, Bel-7402, A549) and normal cell lines (HELF, LO2) were infected separately with rAd-CMV-GFP-hTERTp-ODC as well as with control vector (rAd-CMV-GFP). Luciferase activity assay was performed to determine hTERT promoter activity. Cell growth curves analysis, western blot analysis, flow cytometry analysis and Matrigel invasion assays were performed to assess properties of cell growth and invasiveness. The results showed that there was significant inhibition of ODC expression and cell proliferation in cancer cells treated with rAd-CMV-GFP-hTERTp-ODC compared with cells treated with PBS or rAd-CMV-GFP, and no significant inhibition was detected in normal cells. Our research offers a powerful and safe new therapeutic strategy for cancer targeted treatment.