Abstract: Objective To construct the recombinant adeno-associated viral vector containing human endostatin gene (rAAV-hEndo) and observe the biological activity of the expressed human endostatin in vitro. Methods rAAV-hEndo was prepared using a helper virus-free packaging system. The rAAV viral genome titer was quantified by Taqman real-time PCR, and the endostatin expressed in human umbilical vein endothelial cell line ECV304 was detected by immunofluorescence staining. The effects of endostatin on ECV340 cells were evaluated by MTT cell proliferation assay, cell cycle analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) technique. Results The viral titer of rAAV-hEndo prepared was 2×10¹² vg/ml and the vector had an infection efficiency of 98%. Immunofluorescence staining showed that the humam endostatin protein was expressed mainly in the cytoplasm of ECV304 cells, and the proliferation of the cells was obviously inhibited by the supernatant of rAAV-hEndo, with a inhibition rate of 67.3% 72 h after the addition of the supernatant. ECV304 cells infected with rAAV-hEndo were obviously arrested in G₁ phase, and the G₁-phase cell percentage of treatment group were significantly higher than that of control group [(72.5±4.0)% vs (52.1±2.1)%, P<0.01]. ECV304 cells infected with rAAV-hEndo demonstrated markedly enhanced apoptosis, with a significantly greater apoptotic index than that of the control cells [(32.6±3.2)% vs (4.2±1.9)%, P<0.01]. Conclusion rAAV-hEndo can effectively mediate the expression of biologically active human endostatin, which may facilitate further study of antiangiogenic gene therapy with endostatin for cancers.