Abstract: To investigate the effect of ornithoctonus huwena venom on proliferation of human colon carcinoma cell SW480 in vitro. Methods In this study, human colon carcino SW480 cell lines were cultured in vitro as the experimental model. Human colon carcino SW480 cells were intervened by different concentration solutions of ornithoctonus huwena venom in vitro. MTT chemosensitivity test was used to evaluate the suppression ratio and IC50. Cell morphology changes were observed through the microscope after dyeing by Hoechst 33342. Cells state and quantity were observed through flow cytometry after dyeing by AnnexinⅤ-FITC/PI. Caspase-3 relative activating in cell was evaluated by spectrophotometry. Results MTT chemosensitivity test results showed that human colon carcino SW480 cell quantity was reduced when intervened by 5 mg/ml-100 mg/ml of huwentoxin, with IC50=43.68 mg/ml(48 h). Microscopic observation results showed that SW480 cell quantity was reduced, with various shapes of cells and part of the cells were broken. Flow cytometry results showed that SW480 cell intervened by 10 mg/ml of huwentoxin had the highest rate of apoptosis (57.66±20.44) % and that intervened by 50 mg/ml of huwentoxin had the highest rate of Necrosis (25.36±15.03) %. Activated caspase-3 was detected out when intervened by 100 mg/ml of huwentoxin in SW480 cell. Conclusion Proliferation of human colon carcinoma cell SW480 cell can be suppressed by ornithoctonus huwena venom in vitro .