The vast majority of new HIV infections in male-to-female transmission occurs through semen, where HIV-1 is present in two different forms: as free and as cell-associated virus. In the female lower genital tract, semen mixes with female genital secretions that contain various factors, some of which facilitate or inhibit HIV-1 transmission. Next, HIV-1 crosses the genital epithelia, reaches the regional lymph nodes, and disseminates through the female host. Cervico-vaginal mucosa contains multiple barriers, resulting in a low probability of vaginal transmission. However, in some cases, HIV-1 is able to break these barriers. Although the exact mechanisms of how these barriers function remain unclear, their levels of efficiency against cell-free and cell-associated HIV-1 are different, and both cell-free and cell-associated virions seem to use different strategies to overcome these barriers. Understanding the basic mechanisms of HIV-1 vaginal transmission is required for the development of new antiviral strategies to contain HIV-1 epidemics.