A compelling tool for functional genetics is to silence the expression of multiple related genes concomitantly and reversibly. Such a tool will accelerate the understanding on gene interaction in signaling pathway and the development of comprehensive animal models for human diseases. Multiple gene silencing may be achieved by concurrent expression of multiple miRNA from a Pol II promoter. By comparison, Pol III promoters possess greater capacity to synthesize RNA of high yield and are consisted of compact elements and simple terminators to be convenient for handling. The miRNA-induced gene silencing is a dose-dependent event, and thus, Pol III promoter as a miRNA driver increases the chance to induce phenotypes subsequent to the gene silencing. As a Pol III promoter, endogenous U6 promoter synthesizes small nuclear RNA of high yield and is commonly adapted for miRNA synthesis. Whether U6 promoter is effective to synthesize multiple miRNA in tandem remains to be determined. This study exploited a possibility to express multiple miRNA genes from U6 promoter and also tested the inducibility of varying types of Tet-regulatable U6 promoters. With miR-30a backbone, two miRNA genes were functionally and efficiently expressed from a U6 promoter. The transcriptional activity of Tet-regulatable U6 promoter was tightly regulated by Tetracycline system after sufficient repeats of Tetracycline Operator sequence were introduced within the promoter regions and also between U6 promoter and miRNA gene. This newly developed U6 miRNA system would make multi-gene silencing efficient and reversible.