At present there is no research on the establishment of Chinese zokor’s (Eospalax fontanierii) fibroblast cell lines and their biological characteristics. In this study, we selected 9 kinds of the tissues of Chinese Zokor, a representative animal genetic resource in the Mongolian Plateau, for the experiment. Fibroblast cell lines were successfully established from three tissues, including trachea, lung and xiphoid cartilage, and their biological characteristics were analyzed. The attachment rate and survival rate of the cells were calculated before cryopreservation and after resuscitation, and their chromosome karyotypes were analyzed. The fibroblast-like cells derived from primary culture of trachea, lung and xiphoid cartilage tissues were found in the third or fourth day of culture, and there was more than 90% confluence of adherent cells on the 11th, 16th and 17th day of culture, respectively. The morphology of these three kinds of somatic cells showed fibroblast characteristics (Fig. 1﹣4). The attachment ability of the trachea fibroblast cells was the greatest, and the attachment rate was 98.10% when cultured for 24 h, and the attachment ability of the lung fibroblast cells and xiphoid cartilage fibroblast cells were 95.28% and 94.88% respectively (Fig. 5﹣7). The analysis of the growth curve of fibroblast cells from three sources showed that the proliferative capacity of tracheal fibroblast cells was the best, the pulmonary fibroblast cells were the second and the xiphoid chondro fibroblast cells were the weakest. Tracheal fibroblast cells and pulmonary fibroblast cells entered the logarithmic growth stage (Fig. 8, Fig. 9) on the 6th to 7th day after inoculation. Xiphoid chondro fibroblast cells entered the logarithmic growth stage on day 2﹣3 (Fig. 10). The survival rate of cryopreserved fibroblasts from three different tissue sources decreased significantly. The growth curves of three fibroblasts are all in an “S” type. The multiplication capacity of the trachea fibroblast cells was the strongest, and the maximum proliferation number was 2.435 × 104 per well of a 24-well plate; the maximum proliferation numbers of lung fibroblast cells and xiphoid cartilage fibroblast cells were 1.813 × 104 and 1.521 × 104, respectively. The results of karyotype analysis showed that the chromosome number of fibroblast was 2n = 62 in Chinese Zokor (Fig. 11). To sum up, this study successfully established the fibroblast cells lines from Chinese zokor. The basic biological characteristics of fibroblast cells in this species were revealed, which provided basis for further studying molecular biology and physiological mechanism of Chinese zokor to adapt to the low oxygen and high carbon dioxide tunnel habitat, and provided important experimental materials and references for the further study of its heredity and species evolution.