Abstract: Objective To establish an in vitro culture system for murine lingual epithelial cells (LECs) and provide an experimental basis for investigating epithelial homeostasis, regeneration mechanisms, and oral disease modelling. Methods Primary cells were isolated from mouse tongue tissue using an optimized two-step enzymatic digestion-mechanical dissociation protocol and cultured in Advanced DMEM/F-12 medium without feeder cells. Serial passage characteristics were monitored. Cell identity was confirmed by morphological assessment and immunofluorescent staining for cytokeratin 5 (K5) and cytokeratin 14 (K14). Results Primary LECs were successfully isolated. These cells formed compact colonies by day 3 and exhibited typical cobblestone morphology by days 11-12. The cells could be stably passagable for 3-4 passages before adopting a spindle-shaped phenotype. Immunofluorescence revealed high expression of K5 (96. 22 ± 0. 96%) and K14 (98. 33 ± 1. 12%). Conclusions The enzymatic-mechanical approach described here enables efficient isolation and sustained culture of murine LECs, establishing a technical foundation for subsequent single-cell sequencing studies.