A Yunnan pony ear marginal tissue fibroblast cell line (NYPEM 2/2) was successfully established using the ex2 plant of the ear marginal tissue and then trypsinization the cells from the outgrowth. Observations on cell morphology and dynamic growth, analysis of karyotype and isoenzymes of lactate dehydrogenase and malate dehydrogenase were carried out. The expression of recombinant green fluorescence protein in the cells were also undertaken. The results showed that the population doubling time (PDT) of the cells was 24h; the frequency of cell chromosome number to be 2n=64 was 9219%; the banding patterns of the isozymes of the two enzymes had significant difference between the Yunnan pony ear marginal fibroblast cell line and the fibroblast cell lines of PEM 2/2, MSHEM 2/2 and BLCHE 2/2 derived from the Picdmont bovine ear, Mongolian ovine ear and Beijing local chicken embryo respectively. Tests for the contamination from bacteria, fungi or mycoplasma were negative; the transfection efficiency for the recombinant plasmid was 3213%. This newly established cell line make the Yunnan pony breed, a national important genetic resource preserved at cell level, as well as will provide an effective experimental material for genetic studies on the Yunnan pony