We investigated the inhibitory effect of modified antisense RNA oligonucleotides and cationic liposome-RNA complexes on the repression of hepatitis B virus (HBV) replication and expression. ELISA and quantitative Real-time PCR analysis showed that HBV replication and antigens expression both in pHBV1.3 transduced HepG2 and HepG2.2.15 cells were reduced after treatment with antisense RNA oligonucleotides P-2987, X-60 and X-519. Subsequently, the antisense RNA oligonucleotides and control RNA oligonucleotides were injected via the tail vein into HBV transgenic mice or hydrodynamically injected mice. In the HBV transgenic mice, with the treatment of X-519, HBV pregenomic RNA in the liver decreased by 81%. Cationic liposome further increased the inhibition effectory to 91%. But no significant differences were observed for HBV antigens and HBV DNA copy number in the sera. In acute HBV infection mouse model by hydrodynamic injection, ELISA and quantitative real-time PCR analysis showed that X-519 significantly repressed HBV replication, as measured by HBV pregenomic RNA, antigens expression, and presence of HBV DNA in the sera. Taken together, the synthesized antisense RNA oligonucleotide X-519 repressed HBV replication and antigens expression in vitro and in vivo.