The cytotoxic effects of the crown-of-thorns starfish Acanthaster planci spine venom (ASV) in five cell lines, including human neuroblastoma (SH-SY5Y), human hepatocellular carcinoma (HepG2), human melanoma (A375.S2), human skin fibroblast (CCD-966SK) and mouse macrophage-like cell (RAW264.7) were assayed. The results indicated that ASV showed cytotoxic effects depending on dose in these five cell lines. Specifically, ASV significantly inhibited the proliferation of human melanoma cell line A375.S2 at $10{mu}g/mL$, indicating A. planci spine venom could be utilized as potential chemotherapeutic agent in the treatment of cancer. The cytotoxicity of ASV to human melanoma cell line A375.S2 was relatively well retained at temperature less than $40^{circ}C$, and sharply lost at temperature more than $80^{circ}C$. The cytotoxicity of ASV also sharply lost at extreme pH environments (pH 2 and 12). The cytotoxicity of ASV was attenuated when treated with $Cu^{2+}$ and anti-oxidant N-acetylcysteine. After SDS-PAGE analysis, ASV showed the major protein components ranging from 10 kDa to 37 kDa.