Cancer stem cells (CSCs) are responsible for carcinogenesis, cancer progression, relapse, metastasis and drug resistance. Therefore, the development of drug molecules targeting CSCs plays a vital role in medicinal researching field. However, there are extremely rare molecules that selectively ablate CSCs. The research and development of drugs targeting CSCs is limited due to a lack of anti-CSCs lead compounds. In this study, an anti-CSCs lead compound 35b was discovered, which was derived from the natural chemical scaffold of Symplostatin 4. This compound exhibited a significantly suppressive effect on tumor growth both in vitro and in vivo. Additionally, 35b could significantly reduce the number of melanoma tumor spheres and decrease the percentage of ALDH+ melanoma cells. Further mechanism study illustrated that compound 35b could eliminate the melanoma CSCs by efficiently blocking Wnt/β-catenin signaling pathway. Collectively, our findings would provide a novel chemical scaffold and alternative idea of molecular design for development of anti-CSCs drugs.