Background The stem-cell compartment is the primary target for the accumulation of oncogenic mutations.Overexposure to solar ultraviolet radiation is responsible for the development and progression of ＞90% of skin cancers.Ultraviolet B (UVB) light-induced keratinocyte apoptosis is a strong preventive mechanism against carcinogenesis. The aim of this study was to isolate keratinocytes enriched with putative human epidermal stem cells and to investigate their apoptotic induction by UVB.Methods Keratinocytes enriched with putative human epidermal stem cells were isolated by adherence to collagen Ⅳ and the expressions of β1-integrin and p63 were investigated. Keratinocytes enriched with putative human epidermal stem cells and normal keratinocytes were irradiated with UVB at 0-80 mJ/cm2. The apoptotic response was investigated with phase-contrast microscopy, Hoechst 33342 staining, flow cytometry of annexin V/PI, and procaspase-3 West blotting.Results Keratinocyte enriched with stem cells expressed high levels of p63 protein and β1-integrin and low level of pan-keratin (C11). In comparison to non-irradiated cells, significant apoptosis of keratinocyte enriched with stem cells was found with 40 and 80 mJ/cm2 UVB. However, significant apoptosis of normal keratinocytes was only found for 80 mJ/cm2 UVB.Conclusions Human epidermal stem cells can undergo apoptosis in response to UVB radiation and are more susceptible than other keratinocytes. The method could be used in vitro studies of human epidermal stem cells.