Abstract Cancer stem cells (CSCs) are a small population of cancer cells that possess characteristics associated with normal stem cells such as self-renewal and differentiation into multiple cell types. Additionally, CSCs have been reported to relationship on tumor relapse after cancer therapy. Mitochondrial dysfunction (MD) has also been known to be associated with cancer malignancy. In this report, the relation of MD and CSCs were initially investigated using mitochondrial-depleted rho0 cells derived from Hep3B hepatocellular carcinoma cell line. The Hep3B/rho0 cells showed cancer stem-like phenotypes including tumor initiation, tumor malignancy, stem cell-related signaling, chemotherapies and radiotherapy resistance. These results suggest that mitochondrial dysfunction induces cancer stem-like phenotypes. Taken together, mitochondrial dysfunction plays an important role in the acquisition of cancer stem-like phenotypes and could be a novel therapeutic target to cancer therapy. Citation Format: Yu-Seon Han, Eui-Yeun Yi, Yung-Jin Kim. Mitochondrial dysfunction induces cancer stem-like phenotypes in Hep3B hepatocellular carcinoma cell line. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-22.