We have established a spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) from the mammary gland of a lactating sow and maintained it long-term in culture by continuous subculturing. SI-PMEC cells were maintained for more than 8 months (70 passages) in DMEM/F12 medium supplemented with 10% fetal calf serum, insulin, and hydrocortisone without obvious signs of senescence. When grown at low density on a plastic substratum, SI-PMEC cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobblestone morphology of epithelial cells. The subcultured SI-PMEC cells appeared to proliferate without changes in morphology or growth pattern, with an estimated population doubling time of 20–22 h. With increasing density, SI-PMEC cells organized into lumen-like structures with elongated cells at the margins. SI-PMEC cells from stocks frozen at Passage 30 were subcultured up to 20 times without changes in cell viability, proliferation rate, or morphology. Furthermore, SI-PMEC cells remained immunopositive to an antibody against cytokeratin AE3 and immunonegative to an antibody against a human fibroblast antigen. The SI-PMEC cells could form functional structures resembling ducts, lateral-buds, and alveoli in a Matrigel matrix-dependent manner in vitro. When grown on the Matrigel and stimulated by prolactin, the cells differentiated and formed mammary gland structures and strongly expressed transcripts encoding the milk proteins α-lactalbumin, β-casein, and β-lactoglobulin. Our results indicate that the SI-PMEC cell line can be subpassaged many times and still form functional differentiated secretory structures. To our knowledge, this is the first report of an immortalized mammary epithelial cell line from pig.