The bacterium Chlamydia trachomatis as well as the Human Immunodeficiency virus-1 (HIV-1) are sexually transmitted pathogens; both can infect monocytes/ macrophages and have an obligate intracellular replication cycle. It has been hypothesized that sexually transmitted infections (STIs), including genital Chlamydia, enhance HIV-1 transmission; yet, the underlying biological mechanisms remain unclear. The pathogen¡¯s common biology makes it possible that C. trachomatis and HIV mutually affect each other¡¯s replication cycle during monocyte/macrophage co-infection. To test this hypothesis we used two different bacteria strains in an in vitro HIV co-infection model: Latently virus-infected U1 promonocytes were either inoculated with C. trachomatis serovar D or serovar L2. It was found that serovar D, but not the LGV serovar L2, quickly and significantly reactivates integrated HIV-1 provirus. This reactivation depends on viable bacteria but seemingly neither on a microbial pathogen-associated molecular pattern (PAMP) nor on de novo gene expression by C. trachomatis. Hence, it could be triggered by a preformed protein that the bacterium translocates into the human cell. Because virus production occurs in Chlamydia-infected as well as Chlamydia-free cells and cannot be induced by conditioned culture medium, we propose a cell contact-dependent signal to U1 bystander cells. The HIV reactivation mechanism involves NF-kappaB activation but is independent of eukaryotic de novo protein translation. In summary, the findings might describe a novel Chlamydia-mediated HIV-1 reactivation mechanism from latently infected cells.