To investigate the effect of recombinant adenoassociated virus (rAAV) containing antisense nucleotide against cyclin B1 (ASCCNB1) on osteosarcoma cells in vitro. Methods The rAAV was produced by Cotransfecting HEK293 cell line with the rAAV plasmid pd16-95-AS-CCNB1/neo and AAV/Adenoviral helper plasmid pSH3. The rAAV particles expressing ASCCNB1 were purified and concentrated. Subsequently the virus titer was assayed by dotbloting analysis. Then, human osteosarcoma U2OS cells were infected by rAAV at the pfu of 10SUP>5/SUP>-10SUP>6/SUP>/cell titer. CCNB1 expression was detected by Western blotting and RT-PCR methods, human osteosarcoma U2OS cells growth curve was assayed by MTT, cell cycle and apoptosis were detected by FACS. Results The rAAV-AS-CCNB1 plasmid was successfully constructed and the virus titer is up to 1.0×10SUP>11/SUP> v.g./ml. After infected with rAAV EM>in vitro/EM>, U2OS cells growth was inhibited significantly. Both Western blotting and RT-PCR assay showed that CCNB1 expression of U2OS cells was downregulated by rAAV-AS-CCNB1,and cell apoptosis and GSUB>1/SUB> arrest were observed. Conclusion The recombinant adenoassociated virus