The HIV transactivator protein (Tat) is a multifunctional protein that plays a critical role in viral replication and contributes to several pathological symptoms of HIV‐1 infection, which has the loss of CD4+ T lymphocytes as one of its hallmark features. It has been shown that endoplasmic reticulum (ER) stress, including viral infections, is implicated in cellular dysfunction and cell death through activation of the unfolded protein response (UPR). Here, we demonstrate that the bystander stimulus of Tat on Jurkat cells resulted in time‐dependent overexpression of major UPR markers including ER chaperone BiP, ER stress sensors ATF6, PERK, and IRE1, as well as an increase in levels of downstream mediators eIF2α, ATF4, XBP‐1, and proapoptotic factors CHOP, GADD34, and BIM. This upregulation of UPR mediators was accompanied by decreased cell viability and increased apoptosis as evidenced by blue trypan dye exclusion and flow cytometry assays, respectively. Furthermore, we found that the Tat‐associated apoptosis of Jurkat cells led to the loss of mitochondrial membrane potential and caspase‐12 and ‐3 activation. Taken together, these results suggest that the exposure of HIV‐1 Tat leads to ER stress/UPR triggering which in turn contributes to apoptotic death in Jurkat cells. Significance of the study In the present work, we provide a substantial insight into the link between ER impairment and apoptotic death following a bystander HIV Tat stimulus, revealing an underlying ER‐mediated apoptotic mechanism which could explain the continuous depletion of uninfected CD4+ T lymphocytes observed in HIV‐related disease. Our findings reinforce the relevance of ER stress molecular responses in the course of HIV infection and may afford valuable information for the development of new therapeutic strategies to avoid CD4+ T lymphocyte loss and other disorders induced by circulant Tat.