Non-small cell lung cancer (NSCLC) represents the most common histological type of lung cancer, characterized by a five-year survival rate of 15% and poor prognosis. Accumulating evidence indicates a prominent role of endoplasmic reticulum (ER) stress and the protein kinase RNA-like ER kinase (PERK)-dependent pathway of the unfolded protein response (UPR) in the pathogenesis of NSCLC. Increased expression of downstream targets of PERK was observed in various subtypes of NSCLC, and it was associated with a more aggressive phenotype, high risk of recurrence, and poor prognosis. Therefore, the present study aimed to investigate the biological effect of the selective PERK inhibitor NCI 159456 on A549 NSCLC cells and Human Pulmonary Fibroblasts (HPF) in vitro. Treatment of both normal and ER-stressed A549 cells with NCI 159456 resulted in a significant increase in the mRNA expression level of pro-apoptotic genes like (, (, and ( as well as a decreased level of the anti-apoptotic gene . Cytotoxicity and genotoxicity analyses revealed that NCI 159456 significantly decreased viability and increased DNA damage in A549 cells under normal and ER stress conditions. Caspase-3 and reactive oxygen species (ROS) detection assays demonstrated that NCI 159456 significantly induced apoptosis and increased the ROS level in normal and ER-stressed A549 cells. Importantly, treatment with the inhibitor did not affect substantially normal HPF cells at any used concentration. The results indicate that PERK inhibitors could potentially be applied as a targeted therapy for NSCLC.