Objective: To study the inhibitory effects of antisense oligodeoxynucleotide (ASODN) and its thiophosphate (S-ASODN) on the replication and expression of hepatitis D virus (HDV) in H1δ9 cells and in tupaia body. Methods:After 15mer-ASODN and S-ASODN were synthesized, different concentrations of ASODN and S-ASODN were added to the culture medium of H1δ9 cell line and then HDAg in the supatant of the culture was examined with ELISA and HDV-RNA in the cells determined with dot blot hybridization. Sixteen tupaiae were successfully infected with HDV and mg every other day for 7 times and the control group were injected with sime volume of normal saline. On the 5th, 10th,15th and 20th day after the administration, samples of blood and liver tissues were examined with immunohistochemical method for HDAg and dot blot hybridizaton and in situ hybridization for HDV-RNA. Results: Twenty-four hours after the addition ofa tinal concentration of 6 μmol/L of S-ASODN, the replication of HDV-RNA and the release of HDAg in th e H1δ9 cells were suppressed by 84.5% and 76.14% respectvely. The inhibition was dose-dependent when the final concentrations of 2, 4 and 6 μmol/L of S-ASODN were given. When ASODN and S-ASODN were administered in the same dosage, the inhibition showed no significantly difference between the 2 agents. On the last day of the administration of S-ASODN, 7 out of the 8 tupaiae of the treated group showed negative HDAg and HDV-RNA in the liver tissue while only 1 out of the 8 tupaiae of the congrol group was negative. Ten days after the cessation of drug administration, 3 tupaiae of the treated group and 7 of control were positive of HDAg and HDV-RNA. Conclusion: Our findings show that SASODN efficiently inhibites the replication and expression of HDV gene in H1 δ9 cells and in the body of tupaia, which provides an experimental basis for the anti-HDV application of antisense oligonucleotides.