Macrophages serve as a major reservoir for HIV-1 because a large number of macrophages in the brain and lung are infected with HIV-1 during late stage disease. Recent evidence suggests that those HIV-1-infected macrophages play a key role in contributing to tissue damage in AIDS pathogenesis. Macrophages undergo apoptosis upon HIV-1 infection; however, the mechanisms of this process are not well-defined. Previously, we demonstrated that HIV-1 infection inhibits Akt-1, a critical protein for cell survival of macrophages. In the present study, we investigated the involvement of transcription factor FOXO3a in the regulation of HIV-1-mediated apoptosis in macrophages. HIV-1 infection significantly decreased phosphorylation of FOXO3a and promoted FOXO3a translocation to the nucleus in human monocyte-derived macrophages (MDM). Overexpression of a constitutively active FOXO3a increased DNA fragmentation with decreased cell viability in MDM, whereas a dominant-negative mutant of FOXO3a or small interfering RNA for FOXO3a to knockdown the function of FOXO3a in HIV-1-infected MDM decreased DNA fragmentation and protected macrophages from death in HIV-1-infected MDM. Overexpression of constitutively active Akt-1 increased FOXO3a phosphorylation, suggesting that FOXO3a phosphorylation in human MDM is dependent on Akt-1. We therefore conclude that FOXO3a plays an important role in HIV-1-induced cell death of human macrophage. Understanding the PI3K/Akt-1/FOXO3a pathway and its associated death mechanism in macrophages during HIV-1 infection would lead to identification of potential therapeutic avenues for the treatment of HIV-1 infection.