Abstract: This experiment was conducted to isolate porcine mammary gland epithelial and fibroblast cells from adult porcine mammary gland tissue, and transfect the enhanced green fluorescent protein (EGFP) gene into the cells. Porcine mammary gland epithelial and fibroblast cells were isolated from adult porcine mammary gland tissue using their culture system. EGFP gene was transfected into the cells by lipofection technology. We succeeded in isolating porcine mammary gland epithelial and fibroblast cells from adult porcine mammary gland tissue, and the EGFP gene transfected epithelial and fibroblast cells were obtained. The epithelial cells were short shuttlelike or guboidal, which were connected with each other closely and properly. The cell nucleus were circular or oval which had 24 cores. The fibroblast cells were long shuttlelike. This result shows that porcine epithelial and fibroblast cells could be isolated from adult porcine mammary gland tissue and EGFP could be expressed in the cells.