Hepatic steatosis has been reported to be a risk factor for the development of liver cancer. The underlying mechanism for carcinogenesis remains to be elucidated. It has been postulated that cancer stem cells (CSCs) within tumor tissues are a subset of cells with stem cell properties of self-renewal and undifferentiation. The purpose of this study was to investigate the effects of a saturated fatty acid, palmitate (PA), on CSC-like properties of human hepatoma HepG2 cells. We investigated the effects of PA on HepG2 cells and primary rat hepatocytes (PRH) by exposing them to PA to induce lipid accumulation. Significant fat accumulation was observed by Oil Red O staining in cells exposed to PA, and it was accompanied by significant increase in NFκB (p65) nuclear translocation in HepG2 cells. Notably, PA significantly enhanced the sphere forming ability of HepG2 cells, but not PRH. Furthermore, PA significantly increased stemness gene expressions of Sox2 and Oct4, and sonic hedgehog (Shh) production. Notably, NFκB inhibitors, N-Acetyl-L-cysteine and pyrollidine dithiocarbamate, and a NOX inhibitor, diphenyleneiodonium, significantly attenuated PA-induced sphere forming ability of HepG2 cells. Our results suggest that lipid accumulation may not only induce pro-inflammatory responses in hepatocytes but may also activate CSC-like properties of hepatoma cells through NFκB activation.