Despite semen being the main vector of human immunodeficiency virus (HIV) dissemination worldwide, the origin of the virus in this bodily fluid remains unknown. Of particular significance is the persistence of virus release in the semen of HIV-infected men under antiretroviral therapy, who otherwise show an undetectable blood viral load. It is therefore considered critical to identify the sources of virus shedding in semen for the more efficient control of HIV transmission. A number of studies indirectly suggest that the free viral particles and infected cells contaminating semen are produced within the male genital tract. Our recent findings indicate HIV infection of several semen-producing organs, including the testis (which represents a pharmacological sanctuary for several antiretroviral drugs), thus reinforcing the hypothesis of the local origin of the seminal contamination. Whether one or several of these organs constitute a viral reservoir seeding semen despite antiviral therapies, remains to be determined. In addition, the detection of virus within the testicular germ cells should be taken into account in the context of assisted reproductive techniques using these cells from HIV positive men.