Abstract: This study aimed to establish the isolation and culture method of Min Pig ear skin fibroblasts and to under their biological characteristics. To establish a Min Pig ear skin fibroblasts line, the ear skin tissues of 1-day-old Min Pig were cultivated by explant culture technique and the obtained fibroblasts were purified by enzyme digestion and differential adhesion method. The harvested cells were cryopreserved in liquid nitrogen. The fibroblasts showed a typical ＂S＂ shape growth curve and their survival rate before and after freezing were above 90%. The in vitro isolation and culture method of Min Pig ear skin fibroblasts was successfully established in this study.