Objective To investigate the effects and signal transduction pathway of human laryngeal carcinoma Hep-2 apoptosis induced by the Apoptin gene. Method The recombinant expression plasmid of pVAX1-Apoptin was constructed by inserting Apoptin gene into the downstream of pVAX1 promoter (CMV). The expression of the forgein gene was identified by RT-PCR, western-blot and indirect immunofluorescence (IFA). Recombinant plasmid pVAX1-Apoptin was transfected into Hep-2 cells by application of liposome in vitro. The anti-tumor effects on Hep-2 cells were measured through MTT staining and, the mitochondrial trans-membrane potential (?Ψm) and reactive oxygen species (ROS) were analyzed by flow cytometry. Western blot was used to detect the release of cytochrome c (Cyto c). Caspase-3/9 activities were measured by colorimetric assay. To analysis the in vivo anti-tumor effects of pVAX1-Apoptin, C57BL/6 mice model bearing H22 hepatoma was constructed. After introtumoral injection of pVAX1-Apoptin, H22 solid tumors were significantly suppressed and the survival span was expanded.  Results pVAX1-Apoptin could restrain Hep-2 cells significantly and, had the function of down-regulating ?Ψm, up-regulating ROS, promoting Cyto c release and activating Caspase-3/9. Introtumeral injection of pVAX1-Apoptin could supperss the solid H22 tumor effectively.  Conclusion Cyto c were released from mitochondria by the function of up-regulating ROS of pVAX1-Apoptin. Cyto c triggered Caspase-9 and, after the activation of Caspase-9, downstream apoptotic factors, such as caspase-3, were activated. Eventually, Hep-2 cells were suppressed bymitochondrial pathway apoptosis induced by pVAX1-Apoptin. And pVAX1-Apoptin had the anti-tumor effects on H22 solid tumors.