Tumor tissue is of heterogeneous nature containing a cancer stem cell (CSC) population which is believed to be responsible for failure of current anticancer therapies and which may get further enriched under selective pressure enabling the recurrence of more aggressive forms of the tumor. Therefore, understanding the CSC’s in comparison to normal stem cells is necessary for developing therapies which target the CSC population effectively. CSC’s share some key characteristics with normal stem cells like self-renewal potential, proliferative quiescence and protection from cytotoxic insult. The activation of stemness genes like telomerase in more than 90 % of all cancers ensures endless self-renewal potential. Unlike normal stem cells which have long telomeres, telomeres of cancer cells shorten rapidly during early tumorigenesis and are maintained at a stable length by reactivation of telomerase. We hypothesize that cancer stem cells have a higher telomerase activity than the bulk tumor cell mass for maintaining a critical minimal telomere length necessary for their survival and targeting telomeres and telomerase with the telomere eradicating agent KML001 would specifically erode the CSC subpopulation and would not affect normal stem cells which have long telomeres and slower proliferation rates. We analyzed various prostate cancer cell lines reflecting different prostate cancer progression stages (hormone responsive, hormone unresponsive and drug resistant) for their telomerase and telomere length characteristics as well as for the existence of a CSC subpopulation using side population (SP) assay which is based on the ability of stem cells to express drug and xenobiotics efflux pumps responsible for the resistance to standard chemotherapies. Furthermore, we analyzed the expression of stem cell markers and drug efflux pumps. We have tested the effect of KML001 on cell growth in a standard MTT and tested whether the compound could specifically eradicate the CSC population in the SP assay. Cell growth curves showed that all of the tested prostate cancer cell lines respond with similar sensitivity to KML001 regardless of their drug resistance and cancer stem cell potential. Telomere lengths were shorter in the more progressed prostate cancer cell line DU145 and the taxane resistant cell lines. The taxane resistant cell line had the highest percentage of SP (CSC population) of about 8 and 50% respectively, accompanied with a high level of expression of the drug efflux pump P-glycoprotein. Higher level of telomerase activity was found in the isolated SP (CSC) population. The compound reduced the telomerase expression level and the SP (CSC) population significantly at the IC50 and IC100 concentrations determined before in a standard MTT. We found that the cancer stem cell population is enriched in the drug resistant cell lines and that this population in particular showed the highest telomerase activity necessary for the maintenance of short telomeres in this subpopulation. We suggest that we will detect a decrease in telomere length with KML001 treatment along with the observed reduction in telomerase level which we believe is responsible for the erosion of the cancer stem cell fraction as seen in the SP assay and that this compound may be useful for other types of cancer.