Dye exclusion is a valuable technique to isolate cancer stem cells (CSCs) based on an ability of stem cell to efflux fluorescent DNA-binding dye, especially for tumors without unique surface markers. It has been proven that side population (SP) cells that exclude Hoechst 33342 dye are enriched with stem-like cells in several cancer cell lines. In this study, we isolated and characterized SP cells from human Ewing’s sarcoma cell line SK-ES-1 in vitro. SP cells were detected in SK-ES-1 and comprised 1.2% of total cell population. Only SP cells had the capacity to regenerate both SP and non-SP cells. The proliferation rates were similar between SP and non-SP cells. However, the clonogenicity and invasiveness of SP cells were significantly higher than that of non-SP cells. Further characterization of this SP phenotype presented other properties. SP cells exhibited increased multi-drug resistance and the ATP binding cassette protein (ABC) transporters were up-regulated in SP population. These findings suggest that SP cells derived from Ewing’s sarcoma play the critical role in tumor metastasis and recurrence and might be an ideal target for clinical therapy.