While RNA interference (RNAi) has been widely used to study rotavirus gene function in vitro, the potential therapeutic role for RNAi in vivo has not been explored. To this end, we constructed two recombinant lentiviral vectors containing short hairpin RNA (shRNA) against non-structural protein-4 (NSP4) of bovine rotavirus (BRV), RNAi-351 and RNAi-492. RNAi-351 and RNAi-492 strongly suppressed the transient expression of a FLAG-tagged NSP4 fusion protein in 293T cells. In BRV-susceptible MA104 cells, RNAi-492 more potently silenced NSP4 mRNA than RNAi-351 and combination of the two shRNAs almost completely silenced viral NSP4 gene expression. While 100% of suckling mice exposed to BRV and control shRNA developed severe diarrhoea, no suckling mice exposed to BRV in the presence of RNAi-492 or a combination of RNAi-492/RNAi-351 developed severe diarrhoea, and only 20 and 3.3% developed mild diarrhoea, respectively. In addition, RNAi-492 and RNAi-351 markedly abrogated rotaviral replication in MA104 cells and significantly inhibited BRV replication in mouse pups. These results indicated that shRNAs silencing NSP4 gene had substantial antiviral properties and inhibited replication of BRV in a sequence-specific manner that may have clinical application.