RNA interference (RNAi) has been proved to be a promising strategy to combat Hepatitis B virus (HBV) infection by way of cell culture and animal model studies. In this work, esiRNAs (endoribonuclease-prepared siRNAs) targeting all of the four open reading frames (ORFs) of HBV genome were prepared. In vitro experiment showed that esiHBVP suppressed HBsAg expression most effectively. Its capacity to suppress HBV replication in vivo was then tested. A single dose of 1 microg esiHBVP was able to reduce HBsAg and HBeAg level in the mouse serum by 90 and 89% one day after injection, while the same amount of chemically synthesized siRNA only reduced that by 33 and 45%. Immunostaining of HBcAg showed that esiHBVP inhibited HBcAg expression more potently than chemically synthesized siRNA. Quantification of HBV DNA in the mouse serum showed 1 microg eiHBVP treatment reduced serum HBV DNA copy number to 18% that of the untreated control, while 1 microg siRNA treatment only reduced that to 63%. In conclusion, the data presented here proved that esiRNA is much more efficient in suppressing HBV replication than chemically synthesized siRNA, and it might be a better therapeutic agent to fight against HBV infection.