Background: Cell fate is regulated by the activation and repression of specific genes and microRNAs (miRNAs) are a class of post-translational regulators of gene expression. Psoriasis is a hyperproliferative skin disorder and it has been reported that the stratified epidermis expresses miR135b, which suggests that miR135b is related with epidermal keratinization. Previously, we isolated populations of epidermal cells according to their ability to adhere to type IV collagen (Rapidly adhering (RA) cells; epidermal stem cells, slowly adhering (SA) cells: differentiated cells). Objectives: The effects of miRNAs on epidermal stem cells were investigated. Methods: Type IV collagen-coated dishes were prepared and a subpopulation of those cells was selected by their ability to adhere to type IV collagen-coated dishes. To test the effects of miR135b, cultured keratinocytes were transfected with miR135b mimic. In addition, miR135b was suppressed to study whether inhibition of miR135b can delay differentiation of keratinocytes. At every passage, transfection was repeatedly performed, and a portion of cells were harvested for cell counting, RNA extraction, and protein extraction. The levels of p63, known as a potential stem cell marker, were also checked. To confirm the proliferative effects of ib-miR135b, skin models were constructed using ib-miR135b transfected cells. After 13 days, sections were stained. Finally, to know whether type IV collagen is a target of miR135b, database was searched. It was found that there is a binding site of hsa-miR135b in 3`UTR region of COL4A3 gene. Protective sequence was designed to inhibit binding of miR135b in 3`UTR region of COL4A3 gene. Results: miR135b was significantly increased in SA cells. RT-PCR analysis showed that transfection of ib-miR135b is effective in suppression of miR135b. Cumulative cell numbers showed a big difference. A colony assay showed that ib-miR135b transfected cells showed a higher colony forming ability. At the 9th passage, ib-miR135b transfected cells reached ca 5 X 1010 cells. However, mock-transfected cells expanded only to 1.6 X 1010 cells. In addition, mock- transfected cells showed large vacuoles earlier than ib- miR135b transfected cells. Our observations suggest that the inhibition of miR135b is effective in preventing degenerative changes of epidermal cells. The levels of p63 were also consistently higher in ib-miR135b transfected cells. These results correlate well with the increased proliferative potential of ib-miR135b transfected cells. In skin model study, the number of p63-positive and PCNA-positive cells was higher in the ib-miR135b model. These findings suggested that the inhibition of miR135b may prolong the stemness and proliferative potential of epidermal keratinocytes. Then, niche environment of epidermal cells were examined and the expression of type IV collagen were increased in the ib-miR135b model. Furthermore, the levels of α6 integrin and β1 integrin are increased in the ib-miR135b model. In monolayer culture, expression of COL4A3 was increased by inhibition of miR135b as shown in skin model study. When cells were transfected with miR135b mimic, they became larger. Furthermore, protective sequence of miR135b abolished these effects. RT-PCR analysis also showed that miR135b mimic decreased the expression levels of COL4A3 and protective sequence of miR135b reversed these effects. These findings suggested that type IV collagen is a target of miR135b and important in maintaining the proliferative potential of keratinocytes. Conclusion: Type IV collagen is a target of miR135b and that the inhibition of miR135b may improve the microenvironment and increase the proliferative potential of basal cells. Our results showed the role of miR135b in epidermal keratinocytes and may provide a way to manipulate stem cell fate in the skin.