Abstract: To provide convenience for the research of cells immortalization and viral and bacterial diseases in pig intestine, different dissociation agents and digestion protocol were used to identify the best method to isolate and culture intestinal epithelial cells. The result showed that explant culture used in this study was the best one to isolate epithelial cells from intestine of neonatal swine. The viable epithelial cells have a good capacity of proliferation. IEC attached readily to culture dishes within 22 h, proliferated significantly with 6—7 d, and reached confluence as patches with 10—12 d, lost the characteristic of epithelium after 12 generations. Morphologically features such as polygonal, oval and nonoverlapping cell was found in first 10 generations, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer,and were out of shape in 11th or 12th generation. The isolated cells were identified as cells of epithelial origin by using antibodies against cytokeratine 18. Electron microscopy analysis of the ultrastructural characteristics of primary cultures of swine intestinal epithelial cells showed the presence of a wellorganized brush border and tight junction. Swine intestinal epithelial with a good capacity of proliferation and serial passage can be obtained by explant culture.