The RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally, and causes the degradation of an mRNA containing the same sequence. In this present study, an alternative approach was used to in vitro synthesize enhanced green fluorescent protein (EGFP) specific short interfering RNA (siRNA) using T7 RNA polymerase, and a pEGFP-N1 transfected, human hepatoma cell line Huh-7 derived Huh-7-N cell clone was established. When introduced the siRNA into the EGFP expressing Huh-7-N cells, the EGFP specific siRNA was able to specifically inhibit the expression of EGFP in Huh-7-N. In comparison with that in wild-type Huh-7 or that in Huh-7 co-transfected with pEGFP-N1, the inhibition of EGFP specific siRNA in Huh-7-N cells is more significant and repeatable. It is concluded that a cell clone Huh-7-N, which stably expresses EGFP, has been established, and the in vitro synthesized EGFP siRNA can be used in silencing the EGFP gene expression. This Huh-7-N/EGFP specific siRNA system has been proved reliable and convenient, and can also be applied widely as control in other RNA interference studies. The RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally, and causes the degradation of an mRNA containing the same sequence. In this present study, an alternative approach was used to in vitro synthesize enhanced green fluorescent protein (EGFP ) specific short interfering RNA (siRNA) using T7 RNA polymerase, and a pEGFP-N1 transfected, human hepatoma cell line Huh-7 derived Huh-7 derived Huh-7 derived cell clone was established. N cells, the EGFP specific siRNA was able to specifically inhibit the expression of EGFP in Huh-7-N. In comparison with that in wild-type Huh-7 or that in Huh-7 co-transfected with pEGFP-N1, the inhibition of is EGFP siRNA can be used in silencing the EGFP gene expression. This Huh-7-N / EGFP spec ific siRNA system has been proved reliable and convenient, and can also be applied widely as control in other RNA interference studies.