Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-kappaB (NF-kappaB) activation. By achieving selective, targeted, and early inhibition of NF-kappaB activity, we demonstrate that NF-kappaB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-kappaB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-kappaB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1alpha/vascular endothelial growth factor (HIF-1alpha/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.