In the present study, the effective components of anticancer bioactive peptide-3 (ACBP-3), a novel antitumor agent isolated from goat liver, were analyzed. The CD44 (+) fraction of the human gastric cancer cell line was isolated, and the cells within this fraction that could form spheroid colonies (SCs) were identified as gastric cancer stem cells (GCSCs). Subsequently, the antitumor effect of ACBP-3 on GCSCs was investigated in vitro and in vivo. ACBP-3 dose-dependently decreased the percentage of CD44 (+) cells, suppressed the proliferation of the SC cells and inhibited their clone-forming capacity. Tumor formation from inoculated SC cells took substantially longer when the cells were treated with ACBP-3 in vivo. ACBP-3 alone or in combination with cisplatin suppressed xenograft tumor growth. The antitumor efficacy of cisplatin, when combined with ACBP-3, was enhanced even using half of the normal cisplatin dosage. The combination of cisplatin and ACBP-3 could partially alleviate the body weight loss in the mice. Moreover, treatment with ACBP-3 alone could prevent the body weight loss in the mice. Our study indicated that ACBP-3 inhibited gastric cancer cell growth by suppressing the proliferation of CSCs. ACBP-3 could be a potential CSC-targeting agent, and combined with cisplatin therapy, might be an effective way to clinically treat patients with cancer with a lower dose and reduced toxicity. J. Cell. Biochem. 115: 697–711, 2014. © 2013 Wiley Periodicals, Inc.