Hepatocellular carcinoma (HCC) represents a worldwide health problem. F1F0 ATPase, one of the most unique supermolecule enzymes in the inner mitochondrial membrane, was recently found located also on the plasma membrane of some tumor and epithelial cells. Ecto-F1F0 ATPase might play the major role in maintaining the normal average intracellular pH in those cells relative to tumor acidic extracellular microenviroment. Inhibiting the extracellular F1F0 ATPase on tumor exhibits both antiangiogenic and antitumorigenic activities. We found previously a strain of murine mAb, mAb6F2C4, which binds with beta-catalytic subunit of F1F0 ATPase. Immunofluorescence and flow cytometry assay showed that mAb6F2C4 could bind with plasma membrane of diverse hepatoma cells and HUVEC. Moreover, it could markedly block extracellular ATP generation of SMMC-7721 cells under extracellular acidic condition. In vitro, mAb6F2C4 retarded not only the proliferation and colony forming ability of SMMC-7721 cells, but also the proliferation and tube formation ability of HUVEC. mAb6F2C4 was located on plasma membrane of some hepatoma cells and attenuated dramaticly tumor growth in tumor xenograft models in nude mice. Therefore, we concluded that mAb6F2C4 binding with ecto-beta-subunit of F1F0 ATPase, could inhibit extracellular ATP synthesis and exhibit both antiangiogenic and antitumorigenic activities, which could be further developed for HCC therapy.