The ER stress-mediated apoptosis has been implicated in several neurodegenerative diseases; however, its role in HIV/neuroAIDS remains largely unexplored. The present study was undertaken to assess the involvement and detailed mechanism of IRE1α pathway in HIV-1 gp120-mediated ER stress and its possible involvement in cell death. Various signaling molecules for IRE1α pathway were assessed using SVGA cells, primary astrocytes and gp120 transgenic mice, which demonstrated gp120-mediated increase in phosphorylated JNK, XBP-1 and AP-1 leading to upregulation of CHOP. Furthermore, HIV-1 gp120-mediated activation of IRE1α also increased XBP-1 splicing. The functional consequence of gp120-mediated ER stress was determined via assessment of gp120-mediated cell death using PI staining and MTT assay. The gp120-mediated cell death also involved caspase-9/caspase-3-mediated apoptosis. These findings were confirmed with the help of specific siRNA for IRE1α, JNK, AP-1, BiP and CHOP showing significant reduction in gp120-mediated CHOP expression. Additionally, silencing all the intermediates also reduced the gp120-mediated cell death and caspase-9/caspase-3 activation at differential levels. This study provides ER-stress as a novel therapeutic target in the management of gp120-mediated cell death and possibly in the treatment of neuroAIDS.