Cancer stem cells play an important role in resistance to therapy, invasion, metastasis, and recurrence. CD44 is one of the well-known surface markers for hepatocellular carcinoma and its expression level is related to poor survival and a high recurrence rate. The effect of phenethyl isothiocyanate (PEITC) on SNU449 hepatocellular carcinoma cell line cancer stem cells is not known. The goal of the present study was to investigate whether PEITC regulates the cancer stem cell phenotype of SNU449 cells. Here, cell viability, colony formation, and wound healing assays were performed to determine proliferative and migratory characteristics. Additionally, Caspase 3, CD44, Akt/mTOR, and p38/STAT3 protein expression levels were measured by western blotting. We found that compared to control confluence, gap fill, and migration rate were increased while half gap time was decreased in PEITC-treated cells. Compared to control-treated cells CD44 (3.2 fold) and p-STAT3 (2.44 fold) protein expressions were upregulated in PEITC-treated cells. Results of this study suggest that STAT3-mediated upregulation of CD44 leads to the gain of cancer stem cell phenotype of PEITC-treated SNU449 cells.