The mechanism through which human immunodeficiency virus (HIV) infection causes retinal disease and the loss of vision in AIDS patients remains unknown. The HIV-1 transactivator protein Tat (HIV-1 Tat) plays a pivotal role in the pathogenesis of HIV-1 infection and is often described as pleiotropic at different concentrations. In a previous study, we demonstrated that the HIV-1 Tat protein can disrupt the barrier function of retinal pigment epithelium (RPE) at 100 nM without affecting cell viability. The present study was undertaken to determine if HIV-1 Tat can induce RPE cell death at different concentrations and to determine the mechanism of any observed cell death. The results demonstrated that two RPE cell lines (ARPE-19 and D407) treated with Tat at concentrations of 200 nM and above exhibited reduced growth and apoptosis in a dose- and time-dependent manner. The disruption of mitochondrial outer membrane permeabilisation, the activation of caspase-3/7 and 9, the downregulation of Bcl-2, the upregulation of Bax, and the activation of p38 MAPK, ERK and NF-κB were all observed in HIV-1 Tat-treated RPE cells. When exposed to an inhibitor or transfected with siRNA of p38 MAPK and NF-κB, the level of cell death and deregulation of the expression of the apoptotic protein were attenuated. These effects were not observed with an ERK inhibitor or siRNA. Based on these results, we suggest that the induction of apoptosis by HIV-1 Tat in RPE cells may be mediated by p38 MAPK and NF-κB activation and involve the mitochondrial pathway. Therefore, these pathways may provide clues leading to novel therapeutic approaches for the retinopathy induced by HIV infection.