Trilobatin is a common type of flavonoids compounds derived from Lithocarpus polystachyus Rehd leaves. Previous report suggests that trilobatin was potentially involved in pro-and anticancer, antioxidative and anti-hyperglycemic activities. Here, we investigated the anticancer efficiency of trilobatin on gefitinib resistant lung cancer cells. In this study, MTT assays, EdU incorporation assays, DAPI staining, tumor sphere formation assays, immunofluorescent staining and Western blot analysis were performed to explore the functional role of trilobatin on gefitinib resistant lung cancer cells. The results showed that trilobatin inhibits proliferation of gefitinib resistant lung cancer cells. In addition, the proportions of apoptotic cells were increased along with down-regulated expression levels of Bcl-2 and mitochondrial Cytochrome C while up-regulated Bax, Cleaved Caspase-3, -9, and cytosolic Cytochrome C expression. Moreover, trilobatin decreased tumor sphere formation and expression levels of multiple stemness markers (ALDH1, CD133, Nanog, and ABCG2) in gefitinib resistant lung cancer cells. Furthermore, investigation of the mechanism indicated that trilobatin suppressed activity of NF-κB via decreasing constitutive phosphorylation of NF-κB p65 and IκB-α in gefitinib resistant lung cancer cells. All these results indicate that trilobatin induces apoptosis and attenuates stemness phenotype of gefitinib resistant lung cancer cells, involved with, or partly, the suppression of NF-κB activity.