The present paper aimed to inhibit the expression of 3D polymerase of coxsackievirus B by short hairpin RNAs (shRNAs) with lentiviral vector. Three pairs of RNA interference sequences targeting 3D gene and corresponding control sequences were designed and synthesized. The pair with the highest interference efficiency was screened by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting. The recombinant plasmid pLVTHM-3DshRNA was constructed and co-transfected with psPAX2 and pMD2.G in 293T cells. The packaged lentivirus (titer 5×10⁷ TU) was subjected to functional evaluations in HeLa cells and mice. The inhibition of 3D polymerase expression was observed.