The P18 peptide is a functional fragment of pigment epithelial-derived factor (PEDF), which is an endogenic angiogenesis inhibitor. This study sought to determine the anti-angiogenic bioactivity of the P18 peptide in hepato-cellular carcinoma (HCC) and to elucidate the underlying mechanism. Xenograft tumour growth assays demonstrated the P18 peptide suppressed angiogenesis of HCC in vivo. Wound healing, Transwell and Matrigel-culture assays indicated that the P18 peptide inhibited the cell migration and tube formation of endothelial cells (ECs) in vitro. Cell viability and apoptosis assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry assays suggested that the P18 peptide inhibited angiogenesis by inducing apoptosis of ECs. Angiogenesis- and signal transduction-associated molecules analysed by western blot demonstrated that the P18 peptide targets vascular endothelial cell growth factor receptor 2 (VEGFR2) on ECs. In conclusion, by inhibiting the phosphorylation of VEGFR2, the P18 peptide modulates signalling transduction between VEGF/VEGFR2 and suppresses activation of the PI3K/Akt cascades, leading to an increase in mitochondrial-mediated apoptosis and anti-angiogenic activity. This bioactivity of the P18 peptide may represent a novel therapeutic strategy for the treatment of HCC.