Abstract: Objective　To investigate the effect on in vitro inhibition of the human cytomegalovirus (HCMV) gene replication by RNA interference using different orders of small interfering RNA (siRNA) transfection and HCMV infection. Methods The siRNAs were designed and synthesized according to the sequence of the immediate early (IE) genes of HCMV. The siRNAs were transfected into human embryonic lung fibroblastic (HELF) cells by cationic liposome through different orders including before or after or at the same time as HCMV infection. Simultaneously, the control groups were set including normal control group, negative control group, positive control group and transfection reagent group. The inhibitory effects of siRNAs on HCMV-IE and a positive control gene of GAPDH were examined by fluorescent quantitative PCR and agarose gel electrophoresis. Results The expression level of GAPDH mRNA in positive control group was significantly lower than that in negative control group and transfection reagent group (P<0.05), and the inhibition rate of siRNA was 70.4%. Among different experimental groups of siRNAs transfection before, after or at the same time as HCMV infection and three control groups of negative control group, positive control group and transfection reagent group, the expression level of IE mRNA was significantly different (F=146.93, P=0.000). In the groups of siRNAs transfection before HCMV infection or at the same time as HCMV infection, the expression levels of IE mRNA were significantly lower than those in the group of siRNAs transfection after HCMV infection (P<0.05). Conclusions In vitro, siRNAs can effectively inhibit the expression of target gene and it is better to do siRNA transfection first then followed by HCMV infection. It is suggested that siRNA might have a role in prevention from HCMV infection.