Dimeric bile acid derivatives (DBADs) were developed and tested for their anti-HBV and anti-HDV activities as sodium taurocholate cotransporting polypeptide (NTCP) inhibitors. DBADs exhibited strong and persistent potency of NTCP inhibition, whereas diverse linkers and constitutions showed distinct inhibition features. Motif aa157-165 on NTCP was shown to be a possible binding site of DBADs; therefore, we determined DBADs' selectivity among NTCPs from different species. A cyclized DBAD scaffold DBA-41 exhibited a high affinity to human NTCP (hNTCP). Intraperitoneal administration of DBA-41 to hNTCP-tg mice induced serum total bile acid elevation. DBA-41 may serve as a biological tool to study NTCP physiological function.