Polyamine biosynthesis is controlled primarily by ornithine Decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). To study the inhibitory effects of Ad-ODC-AdoMetDCas on polyamine biosynthesis and esophageal cancer cell apoptosis, adenovirus-mediated gene transduction efficiency was assessed with counting GFP-positive cells using MTT. The malignant phenotype of Eca109 cells was assessed by growth curve. Western blot and HPLC were used to detect ODC and AdoMetDC expression and polyamine content in Eca109 cells. TUNEL was used to analyze cell apoptosis. The change of morphology of apoptotic cells was observed by electron microscope. It was demonstrated approximate 70% of Eca109 cells were infected with Ad-ODC-AdoMetDCas when MOI reached 50. The expression of ODC was inhibited in the infected tumor cells. Ad-ODC-AdoMetDCas could inhibit Eca109 cell growth and invasive ability. TUNEL proved that Ad-ODC-AdoMetDCas can lead to cell apoptosis. Characterized morphology was observed by electron microscope(chromatin condensation, nuclear disintegration, formation of apoptotic bodies). It was suggested Ad-ODC-AdoMetDCas has significant inhibitory effects on esophageal cancer cell proliferation , leads to cell apoptosis and bears therapeutic potential for the treatment of esophageal cancer.