Abstract: We aimed to explore the involvement of Fas-associated death domain protein (FADD) in the inhibitory effects of etoposide (VP16) on the proliferation, migration, and apoptosis of A549 non-small cell lung cancer (NSCLC) cells. FADD knockout (KO) and control A549 cells were constructed using the CRISPR/Cas9 system. The cell counting kit-8 (CCK-8) assay, the scratch wounding assay, and the Annexin V/PI staining-based flow cytometry were used to assess the effect of FADD KO on viability, migration, and apoptosis of A549 cells with or without the presence of etoposide, respectively. The expression pattern of several proteins involved in proliferationraf proto-oncogene serine/threonine-protein kinase (c-Raf) and extracellular signal-regulated kinase of phosphorylation (p-ERK), apoptosisB-cell lymphoma 2 (BCL2), cleaved cysteinyl aspartate specific proteinase 3 (cleaved-caspase-3), and cleaved-caspase-9 and migrationmatrix metalloproteinase 2 (MMP2) was detected by Western blot. We found that FADD KO attenuated proliferation and migration of A549 cells. Consistently, we demonstrated that FADD KO enhanced etoposide-mediated inhibition of proliferation and migration in A549 cells. We further demonstrated that FADD KO obviously enhanced etoposide-mediated apoptosis in A549 cells. For mechanism exploration, we found that etoposide sensitivity enhanced by FADD KO may be partly explained by reduced expression of c-Raf, p-ERK, MMP2, and increased cleavage of caspase-3 and -9. Combined with the Kaplan-Meier (KM) survival curve analysis obtained from the GEPIA database, it is preliminarily judged that patients with high FADD gene levels in lung adenocarcinoma have a poor prognosis. Our study suggests that FADD can be used as a potential biomarker for the treatment of lung adenocarcinoma, providing a personalized treatment plan for the treatment of lung adenocarcinoma.