Objective The aim of this study was to isolate, culture, and characterize the skin fibroblasts from Microtus fortis. MethodsThe primary skin fibroblasts were isolated from Microtus fortis at different ages by trypsin digestion and tissue adherence methods. The growth behavior and the characteristics of Microtus fortis skin fibroblasts(MfSF)cultured in DMEM and 1640 culture medium were studied, respectively. ResultsThe number of cells isolated from 1-3-day old animals was more profit than that from 5-day-old Microtus fortis, isolated by trypsin digestion, and the cell purity was high. The MfSF grew well and attached to the bottle wall with a high purity and good ability of proliferation in the primary passage in DMEM or 1640 culture medium, and the time of confluence was 6-7 days, but the growth became gradually poor in the subsequent passages. HE staining showed that the MfSF had an oval nucleus. It was unsuccessful to isolate and culture MfSF by tissue adherence method in this study. ConclusionA successful and efficient method of isolation and culture of skin fibroblasts from Microtus fortis has been established. It provides a useful method and protocol to obtain MfSF to serve further studies. 