RNA interference (RNAi) is the process of mRNA degradation induced by double-stranded RNA in a sequence-specific manner. Different types of promoters, such as U6, H1, tRNA, and CMV, have been used to control the inhibitory effect of RNAi expression vectors. In the present study, we constructed two shRNA expression vectors, respectively, controlled by tRNA(lys) and CMV enhancer-tRNA(lys) promoters. Compared to the vectors with tRNA(lys) or U6 promoter, the vector with a CMV enhancer-tRNA(lys) promoter silenced pokemon more efficiently on both the mRNA and the protein levels. Meanwhile, the silencing of pokemon inhibited the proliferation of MCF7 cells, but the induction of apoptosis of MCF7 cells was not observed. We conclude that the CMV enhancer-tRNA(lys) promoter may be a powerful tool in driving intracellular expression of shRNA which can efficiently silence targeted gene.