RNA-dependent protein kinase is an interferon-induced, double-stranded (ds), RNA-activated serine/threonine protein kinase involved in the eukaryotic response to viral infection. While PKR also functions in cellular differentiation, growth control and apoptosis, its role in human cancer remains poorly understood. To explore a role for PKR in human cancer, we evaluated PKR expression and function in a series of cancer cell lines from different tumor types. We observed that PKR protein expression is high in various cancer cells and low in normal cells. Knockdown of PKR protein expression by PKR siRNA induced cell death, indicating a PKR-dependent survival pathway under normal growth conditions. Inhibition of PKR signaling using a dominant negative adenoviral PKR mutant (Ad-Delta6PKR) also induced cancer cell apoptosis via a mechanism that blocks activation of AKT-mediated survival while simultaneously inducing ER stress. ER stress-mediated apoptosis was evidenced by unregulated expression of phosphorylated JNK (p-JNK), phosphorylated cJun (p-cJun), and caspase-4 and was significantly reduced in cancer cells treated with JNK and caspase-4 inhibitors. We further demonstrated that inhibition of PKR signaling via either siRNA or Ad-Delta6PKR sensitizes cancer cells to etoposide or cisplatin-mediated cell death. Our results suggest a rationale to develop therapeutic strategies that target PKR signaling in human cancer cells.