Oral cancer is one of the most frequent malignant diseases worldwide, and areca nut is a primary carcinogen causing this cancer in Southeast Asia. Previous studies to examine the effects of this carcinogen often used short-term and high-dose treatment of area nut extract as a research model, which do not recapitulate the conditions of patients with long-term and habitual use of this substance. To approach authentic mechanism of areca nut-induced oral carcinogenesis that occurs in human, we established four isogenic sublines of oral cells which were chronic exposed to areca nut extract. Without eliciting cytotoxicity or senescence, these four sublines cells exhibited significant increase in invasive ability, along with epithelial-mesenchymal transition. These cells also showed resistance to chemotherapeutic drug and irradiation, accompanying with the augmentation of ABCG2 protein efflux and increased ROS clearance. Moreover, these sublines possessed the characteristics of cancer stemness, as demonstrated by enriched CD24−/CD44+ and CD133+ sub-populations, enhanced spheroid cell formation, and induced expressions of pluripotent stemness regulators, including Gp96, Grp78, Slug, Sox9, Snail, and Foxc2. These stemness regulators were further shown up-regulations in oral cancer patients with areca nut-chewing habit, and were statistically correlated with CD44 expression, a stemness marker. In conclusion, our findings suggested that areca nut contributes to oral malignancy through facilitating the conversion of cancer stem cells. This study may further contribute to clinical applications in disease prevention, risk assessment or molecular therapeutics on areca nut- associated diseases. © 2015 Wiley Periodicals, Inc.