Eupatilin is a flavonoid compound isolated from the Artemisia plant. Eupitillin possesses antioxidant as well as potent anticancer properties. In this study, for the first time, we examined the anti-proliferative effects of Eupatilin in human melanoma A375 cells and its ability to induce apoptosis and cell cycle arrest. Eupatilin specifically induced morphological changes in A375 cells and was less toxic to the normal mouse splenocytes. The inhibitory effects of A375 cells were associated with the DNA damage, apoptosis, and cell cycle arrest at G2/M phase in a dose-dependent manner. The apoptotic effects of Eupatilin were further verified by using Annexin V-FITC/propidium iodide staining in flow cytometry. These results suggest that Eupatilin is an effective natural compound that worth further mechanistic and therapeutic studies against human melanoma.