Cancer drug resistance and epithelial-mesenchymal transition (EMT), a critical process of cancer invasion and metastasis, have recently been associated with the existence of cancer stem cells (CSCs). However, there are no appropriate CSC-markers of non-small cell lung cancer (NSCLC)-associated drug resistance and EMT. It is unknown if and how the drug-resistant and EMT phenotypes in NSCLC cells link to specific stemness-related pathways. Here, we found a significant elevated expression of both Oct4 and Nanog in gefitinib-resistant NSCLC cells, which displayed multi-drug resistance (MDR) properties and exhibited EMT phenotype. Ectopic co-expression of Oct4/Nanog empowered NSCLC cells with cancer stem cell properties, including self-renewal, drug resistance, EMT and high tumorigenic capacity. Following molecular mechanism investigation indicated Oct4/Nanog-regulated drug resistance and EMT change through Wnt/β-catenin signaling activation. Moreover, silencing β-catenin abrogated Oct4/Nanog-mediated MDR and EMT process in NSCLC cells. Our findings propose Wnt/β-catenin pathway as a promising therapeutic target for the treatment of progression and metastasis of NSCLC with CSC-like signatures and epithelial-mesenchymal transition phenotype.