The technology involving external guide sequence (EGS) is a new approach for gene inactivation, since the induced endogenous RNase P cleaves the target RNA with high specificity and efficiency. Compared with the conventional designs, a more economical 12 bp optimized EGSs (miniEGSs) was constructed to target human cytomegalovirus (HCMV). To investigate its inhibitory effects, quantitative real-time PCR and Western blot was used to detect the level of UL49 mRNA and protein in a cell line stably express HCMV UL49. The transfection efficiency of FITC labeled miniEGSs was above 97% as assayed by flow cytometry. A significant reduction of UL49 mRNA (50%) was observed by quantitative real-time PCR assay following the miniEGS treatment, and the Western blot analyses confirmed a similar result. The data suggested a potential application to use miniEGSs for HCMV antiviral therapy.