RNA interference (RNAi), which could silence specific gene expression post-transcriptionally, has become a powerful tool for identifying gene function in eukaryotic cells. One important approach of RNA interference is to construct a vector system which can direct the synthesis of small interfering RNAs (siRNA) in cultured mammalian cells. The H1 RNA promoter (374 bp) was cloned from HepG2 genome DNA. Two RNAi vector systems, pSL and pESL which has an EGFP gene, were constructed and used to knock down the expression of p53 gene. Then, the mRNA and protein expression of p53 gene were detected by semi-quantitative RT-PCR and Western blotting after the RNAi plasmids were transiently transfected to the HepG2 cells. It turned out that the RNAi efficiency of pSL and pESL are much higher than that of pSilencerTM 3.1-H1 hygro RNAi vetor. Therefore, the data suggested that pSL and pESL RNAi vector systems, which could suppress specific gene expression with high efficiency, are new useful tools to identify gene functions in cultured mammalian cells.