Interleukin-33 (IL-33) has a variety of biological activities in different pathological models. However, the underlying effects of IL-33 on hepatocellular carcinoma (HCC) have not been fully elucidated. Therefore, we focused on investigating the biological effects of IL-33 on HCC stemness expansion. IL-33 expression in clinical tissue specimens were evaluated using immunohistochemical technology. Western blotting, flow cytometry, and quantitative real-time polymerase chain reaction were used to detect protein expressions in HCC cell lines. ST2 expression was downregulated by utilizing the synthetic siRNA sequence that specifically targets ST2. The transfection of ST2 siRNAs and control siRNAs into HCC cells was performed with Lipofectamine RNAi MAX (Life Technologies) according to the manufacturer's protocol. Our results demonstrated that IL-33 is expressed both in cancer cells and stromal cells of the HCC microenvironment, and that IL-33 expression in cancer cells, but not in stromal cells, was negatively associated with survival of HCC patients. IL-33 promotes HCC stemness expansion, including upregulating core stem cell gene expression, inducing cell sphere formation and preventing chemotherapy-induced apoptosis in vitro. Mechanistically, IL-33 binds to its receptor ST2 and induces phosphorylation of c-Jun N-terminal kinase activation (JNK), which leads to HCC stemness expansion. IL-33/ST2 signaling might be potential prognostic markers and therapeutic targets for HCC patients.