We developed several adenoviral vectors designed to target MDA-7 expression to different subcellular compartments [endoplasmic reticulum (ER), mitochondria, nucleus, and cytosol] and evaluated their ability to enhance apoptosis. Adenoviral ER-targeted mda-7/interleukin-24 vector (Ad-ER-mda7) selectively and effectively inhibited the growth and proliferation of lung (A549 and H1299) and esophageal (Seg1 and Bic1) cancer cells by enhancing cell killing. Both Ad-mda7 and Ad-ER-mda7 activated a novel pathway of ER stress-induced apoptosis characterized by unregulated expression of phosphorylated JNK, phosphorylated c-Jun, and phosphorylated RNA-dependent protein kinase. Caspase-4 activation mediated Ad-mda7- and Ad-ER-mda7-induced cell death. In addition, Ad-mda7- and Ad-ER-mda7-mediated growth inhibition correlated with activation of ER molecular markers RNA-dependent protein kinase and JNK both in vitro (in Ad-mda7- or Ad-ER-mda7-treated lung cancer cells) and in vivo. These findings suggest that vectors targeting the ER (Ad-ER-mda7) may be more effective in cancer gene therapy possibly through more effective induction or ER stress pathways.