The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2′-F substituted RNA aptamers that bind to the HIV-1_BaL gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer–siRNA chimeras. The aptamers and aptamer–siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a ‘sticky’ sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.