The RNA interference pathway is a mechanism to regulate gene expression that acts in mammalian antiviral immunity as a complement of interferon- and cytokine-based innate immunity. RNA interference has been proposed as an ancient mechanism against viruses since several components of this system show an effect against viral replication. In fact, protein effectors of this pathway, as well as synthesized microRNA, act against HIV-1, exerting a partial control over HIV-1 latency and replication. Conversely, HIV-1 may counteract this antiviral cell response through two major lines of attack: first, its main regulator Tat suppresses the cellular RNA interference pathway; and second, the virus synthesizes viral microRNA that alter specific cellular functions to enhance HIV-1 replication. As a result of this complex interaction, the microRNA profile in an HIV-1-infected cell is deeply modified. One key application of all this knowledge is the development of pharmacological treatment since microRNA expression can be manipulated and artificial small interference RNA can be delivered into the infected cell to inhibit viral replication. This strategy, combined with current antiretroviral therapy, could be valuable in controlling HIV-1 replication in infected cells.