In our genomes there are thousands of copies of human endogenous retroviruses (HERVs) originated from the integration of exogenous retroviruses that infected germ line cells millions of years ago, and currently an altered expression of this elements has been associated to the onset, progression and acquisition of aggressiveness features of many cancers. The transcriptional reactivation of HERVs is mainly an effect of their responsiveness to some factors in cell microenvironment, such as nutrients, hormones and cytokines. We have already demonstrated that, under pressure of microenvironmental changes, HERV-K (HML-2) activation is required to maintain human melanoma cell plasticity and CD133+ cancer stem cells survival. In the present study, the transcriptional activity of HERV-K (HML-2), HERV-H, CD133 and the embryonic transcription factors OCT4, NANOG and SOX2 was evaluated during the in vitro treatment with antiretroviral drugs in cells from melanoma, liver and lung cancers exposed to microenvironmental changes. The exposure to stem cell medium induced a phenotype switching with the generation of sphere-like aggregates, characterized by the concomitant increase of HERV-K (HML-2) and HERV-H, CD133 and embryonic genes transcriptional activity. Although with heterogenic response among the different cell lines, the in vitro treatment with antiretroviral drugs affected HERVs transcriptional activity in parallel with the reduction of CD133 and embryonic genes expression, clonogenic activity and cell growth, accompanied by the induction of apoptosis. The responsiveness to antiretroviral drugs treatment of cancer cells with stemness features and expressing HERVs suggests the use of these drugs as innovative approach to treat aggressive tumours in combination with chemotherapeutic/radiotherapy regimens.