A Texel sheep ear marginal tissue fibroblast cell line (named TSF19) was successfully established by using a primary explant technique and cell cryoconservation technology. TSF19 cells were adherent, with a population doubling time of 24.9 h. Chromosome analysis showed that >90% of cells were diploid prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the TSF19 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, or mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pECFP-N1, pDsRed1-N1, and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 21.8% and 46.5%. This newly established cell line will not only preserve the genetic resources of the important Texel sheep at the cell level but will also provide a valuable resource for genomic, postgenomic, somatic cloning research.