RNA polymerase Ⅲ is an essential enzyme in eukaryotes for synthesis of tRNA, 5S rRNA, and other small nuclear and cytoplasmic RNAs.Thus, RNA polymerase Ⅲ promoters are often used in small hairpin RNA (shRNA) expression.In this study, the porcine H 1, U6, and 7SK RNA polymerase Ⅲ type promoters were cloned into a pcDNA3.1(+) expression vector containing a shRNA sequence targeting enhanced green fluorescent protein (EGFP).PK and DF-1 cells were cotransfected with a combination of one interference expression vector and the EGFP expression vector, pEGFP-N1.The average fluorescence intensity of EGFP in transfected cells was measured by fluorescence microscopy and flow cytometry.Real-time PCR was used to detect expressed shRNAs and the relative expression of EGFP, to confirm the activity of the promoters.The results showed that the porcine 7SK promoter is stronger than the U6 promoter, which is in turn stronger than porcine H1.This study confirms that the porcine H1 promoter is effective for expression of shRNA, and may be an excellent tool to knock down gene expression in pigs for functional genomics studies.The results also lay a foundation for the development of porcine RNAi technology and transgenic porcine research.