ABC transporters are often found to be inherently expressed in a wide variety of stem cells, where they provide improved protection from toxins. We found a subpopulation of human melanoma cells expressing multidrug-resistance gene product 1 (MDR1). This fraction co-expresses the ABC transporters, ABCB5 and ABCC2 in addition to the stem cell markers, nanog and human telomerase reverse transcriptase (hTERT). The clonogenicity and self-renewal capacity of MDR1(+) melanoma cells were investigated in single cell settings using the limiting dilution assay. We found that the MDR1(+) cells, isolated by FACS sorting, demonstrated a higher self-renewal capacity than the MDR1(-) fraction, a key stem cell feature. Moreover, MDR1(+) cells had higher ability to form spheres in low attachment conditions, a hallmark of cancer. In conclusion, these novel findings imply that the MDR1(+) cells represent melanoma stem cells and thus should be considered as a unique cellular target for future anti-melanoma therapies.