Sanguinarine, an alkaloid present in various medicinal plants, has been associated with detrimental effects on different types of cancer cells. The aim of our study was to analyze the cytotoxic effects of sanguinarine on C32 human amelanotic melanoma cells. The cells were exposed to various concentrations of sanguinarine (0.5, 1, and 2 µM) for 24 h and displayed a dose-dependent decrease in cell viability; the sanguinarine-induced oxidative stress in C32 cells affected the activity of antioxidant enzymes and generated lipid peroxidation. Moreover, we observed an enhanced expression of heat shock proteins 60, 70, and 90 in response to the stress exerted by this alkaloid. The increased p53 protein expression and Bax/Bcl-2 ratio revealed the susceptibility of C32 cells to apoptosis. Our study demonstrated that sanguinarine induced cytotoxicity in melanoma cells that ultimately resulted in cell death. These results emphasize the antigrowth efficiency of sanguinarine against amelanotic melanoma cells, but further research is needed in order to obtain more potent antitumor effects of this alkaloid.