Background RNA interference based therapeutic approaches hold promise for the treatment of patients chronically infected with hepatitis B virus (HBV). To conquer HBV infection, long-term suppression of target transcripts in all hepatocytes without toxic effects may be required. The present study explored gene-deleted adenoviral vectors (GD-AdV) lacking all viral coding sequences for delivery of the previously described short hairpin RNA (shRNA) HBVU6no.2, which was demonstrated to result in post-transcriptional knockdown of HBV transcripts.