OBJECTIVE The aim of this study was to establish a stable in vitro culture system for keratinocytes obtained from oral lichen planus (OLP) lesions and evaluate cultured keratinocyte characteristics including cell morphology, ultrastructure, and expression of biomarkers. METHODS OLP mucosa (histopathologically confirmed) was collected and cells isolated using the cold enzyme digestion method. Primary culture and serial passage were performed on serum-free keratinocyte medium. Morphological changes of cells were evaluated via inverted phase contrast microscopy, and cellular ultrastructure was observed by electron microscopy. Indirect immunofluorescence was used to detect expression of keratin and nuclear factor-kappaB (NF-κB). RESULTS OLP type I keratinocytes was successfully cultured in vitro in serum-free medium. Cellular morphology was typically polygonal during the growth phase. Cells could be passaged continuously for five to six generations without losing viability. Transmission electron microscopy showed large nuclei and multiple vacuoles in the cultured cells consistent with histopathological features of OLP keratinocytes. Indirect immunofluorescence staining was positive for keratin and NF-κB. CONCLUSIONS This study established that human OLP kera-tinocytes can be successfully cultured cells with histopathologic features and biomarker expression consistent with OLP type I keratinocytes. CONCLUSIONS This culture system lays a foundation for the establishment of human OLP cell model in vitro.