Sorafenib is a systemic chemotherapeutic agent for advanced hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the anticancer effect of sorafenib in cancer stem cell‑like cells, such as side population (SP) cells, in HCC and to analyze the signaling pathway for drug‑resistance. To evaluate the anticancer effects of sorafenib, Huh7 and Huh‑BAT cells were treated with sorafenib, fluorouracil (5‑FU), and sorafenib plus 5‑FU. These cells were examined for growth rates, the SP fraction, sphere‑forming efficacy and expression of c‑Jun N‑terminal kinase (JNK) signaling molecules. Sorafenib and 5‑FU treatment decreased growth rates in Huh7 and Huh‑BAT cells; however, the treatments exerted different effects in SP cells and on the expression levels of JNK signaling molecules. Treatment with 5‑FU increased the SP cell number and upregulated the expression of JNK signaling molecules. By contrast, sorafenib decreased the SP cell number and downregulated the expression of JNK signaling molecules. No significant differences in sphere‑forming efficacy were observed subsequent to 5‑FU and sorafenib treatment in Huh7 and Huh‑BAT cells. These results indicate that sorafenib exerted anticancer effects in HCC and SP cells by targeting JNK signaling.