Exosomes are membranous, extracellular nanovesicles secreted by a variety of cell types that contain RNA and protein cargos derived from cellular cytoplasm of cells. Exosomes play a role in cellular signaling, antigen presentation, dysregulation of immune response, and microbial pathogenesis. Previous research from our laboratory has shown that exosomes from healthy human semen suppress HIV-1 replication and impair progeny virus fitness, thus inhibiting viral propagation. Our aim in this study is to determine the effect of semen exosomes (SE) on (i) HIV-1 latency and (ii) activated latent viruses. Using vehicle treated U1 cells (U937 monocytic cells harboring latent HIV-1) we show that SE increased the level of HIV-1 proviral DNA in latent cells on day 2. However, SE significantly decreased proviral DNA in these cells by day 10. In contrast to the latent cells, SE decreased proviral DNA on both days 2 and 10 in cells following stimulation with PMA. Analysis of progeny fitness reveal that prolonged exposure of latent viruses to SE results in significant decrease in progeny fitness as determined by reduced viral reverse transcriptase (RT) activity. Upon activation, progeny fitness increased both in SE and vehicle treated cells over time. A head to head comparison of progeny generated in the presence and absence of SE on days 2 and 10 confirms that SE reduced HIV-1 RT activity. This observation is consistent with SE-mediated impairment of HIV progeny fitness. These data suggest that the anti-HIV-1 factor in SE could be harnessed as a tool for the “shock and kill” approach of eliminating HIV-1 from the population.