Despite their initial effectiveness in the treatment of melanoma, chemotherapeutic agents are ultimately futile against this most aggressive form of skin cancer, and patients inevitably succumb to the disease. One of the mechanisms by which residual melanoma cells become chemoresistant is via the decreased efficiency of chemotherapeutics through the action of ATP-binding cassette (ABC) proteins that are variably expressed by the tumor cells. The clinical relevance of the ABC transporters in the context of cancer is paramount. Inhibitors of these transporters have been shown to increase the efficacy of standard therapy in experimental systems. Their clinical application requires better understanding of the role individual transporters play in the mechanism and the development of more specific inhibitors with minimal off target effects. ABC transporters in tumor cells have been shown to confer multidrug resistance in many solid tumors. However, their role in melanomas is far from clear. Here, we prospectively identify ABCB8 as a specific and major player in the chemoresistance of several melanoma cell lines. ABCB8 knockdown with shRNA reduced doxorubicin resistance approximately 3- to 4-fold in these cells. Furthermore, we show that this reversal is specific to doxorubicin and not to other commonly used chemotherapeutics. Our results also provide evidence that ABCB8 conferred resistance through the protection of mitochondrial DNA from doxorubicin-induced DNA damage.