We have constructed four different RNA polymerase III (Pol III)-based expression vectors, containing H1 or U6 promoters from human and mouse, which enable the endogenous production of small RNA transcripts for gene silencing applications. In addition, to facilitate the selection of recombinant clones, we have further improved these vectors by constructing a stuffer of puromycin resistance gene (Puro(r)) between ClaI and HindIII sites, which makes the preparation of vectors easy for rapid and efficient cloning of targeting sequences. A comparative analysis of the silencing efficiency between shRNA, sense-RNA, antisense-RNA, and siRNA showed that both the shRNA and siRNA, but not the sense-RNA and antisense-RNA, dramatically inhibit the targeting gene firefly luciferase activity in mammalian cells. However, there were no significant differences in the inhibition of firefly luciferase expression by shRNA and siRNA expressed from these DNA vectors. In summary, these improved DNA vector-based RNAi systems should provide a simple, convenient, and efficient cloning strategy for studying gene functions in mammalian cells.