Nine different DNA enzymes(DzV3-n,n=1-9)targeting the V3 loop region of HIV-1 HXB2 were synthesized. One of those, DzV3-9, efficiently cleaved the target in the conserved sequence in the RNA transcript in vitro.DzV3-9 was stable in the cells and inhibited replication of both NL432 and SF162 strains in U87 cells expressing CD4 and co-receptors. The inhibitory effect of DNAzyme on incoming HIV-1 was also demonstrated with pseudotype virions generated by NL432-based luciferase reporter genes.Thus,an efficient,stable DNAzyme against a functionally important region of HIV-1 was identified,and it may be useful for prevention of HIV-1 infection.