The aim of this study is to try to develop an RNA interference (RNAi) approach to human cytomegalovirus (HCMV) therapy by inhibition of viral DNA polymerase (product of the UL54 gene) expression in vitro. The fusion protein expression plasmid pEGFP-UL54 was constructed and cotransfected into AD293 cells with the lentiviral vector pGCL-GFP expressing small hairpin RNA (shRNA) especially targeting the UL54 gene. At 24, 48, and 72 h posttransfection, expression of the fusion protein was detected by fluorescence microscopy and Western blot. The level of UL54 mRNA was semiquantitated by reverse transcription polymerase chain reaction. It was found that, compared with the negative control, expression of the EGFP-UL54 fusion protein was efficiently inhibited and the mRNA level of the UL54 gene decreased significantly in cells introduced with a shRNA producing plasmid called pGCL-UL54-1479. However, the plasmid pGCL-UL54-2884 had no significant inhibitive effect on protein expression or mRNA level. It may be concluded that introduction of shRNA targeting UL54 gene of HCMV could specifically inhibit the expression of viral DNA polymerase in vitro. Nucleotides 1479-1499 of the UL54 gene seems to be an effective target site of RNAi.