Background: Gene silencing mediated by small interfering RNA (siRNA) has become a powerful biological tool for the regulation of gene expression. For the synthesis of siRNA by vector-based expression systems, several mammalian small nuclear RNA (snRNA) promoters have been cloned and shown different transcriptional efficiencies. Results: In this study, we identified a sheep 7SK snRNA (s7SK) promoter based on the highly conserved polymerase III promoter elements. Promoter activity was measured by promoter-driven shRNA expression to suppress expression of an exogenous reporter gene and endogenous sheep gene. Conclusions: The knock down assay demonstrated that the s7SK induced more stronger inhibition effect than human U6 and H1 promoters. The use of this native sheep 7SK promoter for shRNA expressionis an important component for development of RNAi-based gene therapy and production of transgenic animals in sheep species.