It has been reported that human cytomegalovirus (HCMV) miR-US25-2 reduces DNA viral replication including HCMV. However, the mechanism remains unknown. In our study, eukaryotic translation initiation factor 4A1 (eIF4A1) was identified to be a direct target of miR-US25-2-3p. Small interfering RNA (siRNA) and miR-US25-2-3p mediated eIF4A1 knockdown experiments revealed that high level of miR-US25-2-3p in MRC-5 cells decreased HCMV and host genomic DNA synthesis, and inhibited cap-dependent translation and host cell proliferation. However, eIF4A1 up-regulation induced by miR-US25-2-3p inhibitor increased HCMV copy number. Therefore, the over-expression of miR-US25-2-3p and consequent lower expression of eIF4A1 may contribute to the inhibition of HCMV replication.