Reverse Transcriptase (RT) activity is historically associated with the replication of infectious retroviruses. Cellular RT-coding genes have subsequently been identified in eukaryotic genomes. These genes are harbored within retrotransposable elements (retrotransposons and endogenous retroviruses), mobile DNA sequences characterized by the ability to integrate in mammalian genomes through RNA intermediates. Retrotransposition is mediated by an RT activity that catalyzes the reverse transcription of RNA into cDNA copies. A vast body of correlative evidence links up-regulated RT activity to cell systems with a high proliferative potential and low differentiation level, including embryonic tissues and tumors. In contrast, RT is silenced, or expressed at low levels, in differentiated cells. In recent work, we have used non-nucleosidic RT inhibitors widely employed to treat HIV infection and we have observed that these molecules exert a powerful cytostatic and differentiating activity in several models of human cancers both in vitro and in vivo, associated with the inhibition of endogenous RT activity. This review addresses the potential role of RT inhibitors as new anticancer therapeutic drugs. Based on preclinical observations, we also discuss the working hypothesis that the differentiating activity of RT inhibitors may re-establish or improve the efficacy of conventional treatments in specific conditions, such as hormone-refractory prostate carcinoma, anaplastic thyroid tumors and hematological malignancies. These novel findings strongly support the need for clinical trials to test the anti-tumor activity of RT inhibitors in specific malignancies.