Abstract: In this study, the replication of rabies virus (RV) was inhibited by RNA interference in vitro experimentally in order to accumulate essential data of the application of RNAi in the researches of RV genomic function and the postexposure treatment of rabies. Targeting N gene of RV, four shRNA expression plasmids were designed and constructed based on the vector pRNATU6.3Hygro which expresses fusion protein of green fluorescent protein as a report gene. Four cell strains (BHKN1, BHKN2, BHKN3, BHKN4) expressing the short hairpin RNAs(shRNA) were obtained after the plasmids were transfected into the BHK21 cell line and screened under the pressure of Hygromycin B (300 μg·mL-1). These cell strains in a 24well tissue culture cluster were infected by 100TCID50 rabies virus CVS11 strain respectively, and 24, 48, 72, 96 h later, the virus replication were detected and evaluated by realtime RTPCR, 50% tissue culture infective dose(TCID50), directed immunofluorescence assay (DFA) and Western blot. At the 48th hour postinfection, the results of the realtime RTPCR indicated that four strains inhibited RV replication in various degree, in which BHKN2, BHKN1 had resulted in a better inhibitory effect(99%, 67.72%) than BHKN3, BHKN4(38.59%, 11.33%). TCID50 analysis showed that the virus titer of the cell strains were reduced by 24, 96.2, 2.14, 1.1 folds respectively compared with that of empty vector control. Inhibition ratios were 31%, 1%, 54%, 82% respectively compared with empty vector control detected by DFA. The Western blot results showed that the RV N protein bands were significantly weakened in BHKN2, BHKN1 cell strains. These findings suggested that the levels of replication of RV were dramatically decreased by BHKN1 and BHKN2 targeting the regions starting at positions 489 and 701 of rabies virus nucleoprotein gene. This study provided an alternative to block the infection of RV and to research the functions of RV genome.