In the last decade, many of the venom-based bioactive peptides demonstrated with a broad and diverse spectrum of pharmacological activities, which help to enlarging the current drug-screening library for searching of new specific biomarkers and novel prototype drugs for diagnosis of diseases such as cancer. Moreover, it paved a new insight to overcome the current drug discovery problems, which including drug resistance problem, side effect and so on. Here we report the discovery of a panel of novel venom-based peptides, which can significantly inhibit the growth of human colon cancer cells. Through our in-house developed high throughput screening techniques and bioinformatics analysis, we successfully isolated eight novel peptides from scorpion venoms with anticancer activities. We performed a series of anti-proliferative assays to demonstrate that these peptides could inhibit the growth of a broad spectrum of tumor cells, especially in human colon cancer cell line; while almost have no effect on normal human epithelial cells. Haemolysis assay was also performed to show our peptides do not cause any harm on normal human red blood cells, which point to the direction of bringing those peptides into the preclinic trial studies. Before the in vivo studies, we are currently investigating the anticancer mechanisms and involved signal pathways of these peptides. LDH assay and real-time live cell analysis were performed, and from those data it indicates that the peptides might actually work by inhibiting the cancer cell growth instead of disrupt their cell membrane directly. Flow cytometry and western bolt results also showed that the peptides have a significant G1 phase cell cycle arrest effect through inhibiting cyclin dependent kinases CDK4 and up-regulating the expression of cell cycle protein regulator Cyclin D3, p27, p21. Moreover, from our current data, it demonstrated that our peptides wouldn’t induce any apoptosis during the cell cycle process. With the solid anticancer mechanism/pathway data, the potential anticancer peptides to target in vivo malignant tumor will be evaluated in cancer xenograft animal model to demonstrate the growth inhibitor activity of our lead peptides. In summary, our study demonstrate that our peptides can inhibit tumor cell growth via cell-cycle arrest, and almost have no cytotoxicity on human epithelial cells, which could lead our lead peptides to further clinical investigation for the new strategy of peptide mono-/combination therapy.