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 a subset 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. Our recent findings indicate HIV infection of several semen-producing organs, including the testis (which represents a pharmacological sanctuary for several antiretroviral drugs). This reinforces phylogenetic observations suggesting that the free viral particles and infected cells contaminating semen are produced within the male genital tract. The fact that HIV replicates within the male genital organs raises several questions: Is one or several of the male genital tract organs responsible for the persistence of HIV in semen despite efficient antiviral therapies? What is the nature of HIV interactions with spermatozoa and testicular germ cells? Recent results established that semen from HIV negative men modifies HIV infectivity: does the seminal fluid from HIV+ men enhance or inhibit the efficiency of HIV sexual transmission?