The number of genetically modified pigs has dramatically increased in recent years. However, common obstacles have been lack of desired phenotypes. This study describes the identification and characterization of the Nandrolone phenylpropionate porcine Rosa26 promoter and locus, and demonstrates the activities of ubiquitous and tissue-specific promoters at the locus, providing an important advancement for genetical modification inpig. Consistent with previous report, we confirmed that porcine Rosa26 gene located on porcine chromosome 13 and obtained a 646 bp-long transcript from the pRosa26 locus, which is longer than the one Li et al. descrided. The termination signal was found in the 39 end of our obtained transcript, suggesting that at least we get the full-length of one transcript variant of pRosa26. Unlike the highly conserved promoter region of Rosa26 gene between mouse and pig, the porcine transcript exhibited low homology with that of mouse and rat, indicating the function of Rosa26 gene may be non-conservative. Actually, little is know of the function of Rosa26. It possibly works to regulate the Venlafaxine HCl expression of ThumpD3. In mouse, human and rat, Rosa26 overlaps with the ThumpD3 gene, which is positioned in the reverse orientation downstream of Rosa26, however, there are two ThumpD3 genes in pig, and pRosa26 locates in the reverse orientation upstream of one ThumpD3 gene and in the same orientation upstream of another ThumpD3 gene. pRosa26 promoter, conserving functions of its homologues in mouse, human and rat, exhibited a wide activity in variety of adult tissues. Furthermore, pR26 could drive transgene expression in a high and stable manner, unlike CMV, a ubiquitous promoter, which was hypermethylated resulting in a low activity, indicating the porcine endogenous promoter is not rejected in the porcine cellular contexts by epigenetic silencing. We believe that genetically modified pigs will profit from the pRosa26 promoter. Rosa26 locus is an ideal site for ubiquitous expression of transgene. In the study, we targeted EF1a-GFP and MyoP-Fst expression cassettes into the pRosa26 locus.