A selective epidermal growth factor receptor inhibitor, Gefitinib, has been clinically demonstrated to be effective for certain cancer cell types including lung cancer. Our previous study indicated that Gefitinib induced Fas/caspase-dependent apoptosis in human lung adenocarcinoma A549 cells. However, the pathway relaying the signals of Gefitinib-induced cell death has not been fully elucidated. Loss of normal function of p53 facilitates the development of neoplastic lesions and possibly contributes to the development of resistance to chemotherapy. Thus, the current study was designed to examine the role of p53 in Gefitinib-induced apoptosis. Incubation of human lung adenocarcinoma A549 cells with 25 μM Gefitinib resulted in phosphorylation and activation of p53 such as enhanced DNA binding activity, which was accompanied by the upregulation of PUMA (p53 upregulated modulator of apoptosis) and Fas, and downregulation of survivin and XIAP (X-linked inhibitor of apoptosis protein). The Gefitinib-mediated Fas, PUMA, survivin, XIAP regulation and subsequent apoptosis were significantly inhibited in stable p53-shRNA transfectants. Similarly, H1299/p53 cells were more sensitive to Gefitinib compared to H1299 cells in clonogenic survival assay. This event was accompanied by p53 phosphorylation, as well as Fas, PUMA, survivin, and XIAP modulation. Collectively, the results support an important role of p53 in Gefitinib-induced apoptosis in human lung cancer cells. p53 may induce apoptosis through the regulation of apoptotic (Fas and PUMA) and anti-apoptotic (XIAP and survivin) genes. Our studies not only pave a way to the understanding of pharmacological mechanisms of Gefitinib, but also implicate for the necessity to prescreen p53 expression level before clinical application of Gefitinib in human cancer therapy.