During infection with human cytomegalovirus (HCMV), overexpression of hcmv‐miR‐US33 can inhibit the lytic viral replication and down‐regulate US29 mRNA. However, it remains unknown whether inhibition of viral replication by miR‐US33 is mediated by down‐regulation of expression of US29 or another host gene. Here, we identified the host gene Syntaxin3 (STX3) to be a direct target of hcmv‐miR‐US33‐5p using Hybrid‐PCR and luciferase‐reporter assays. It was further demonstrated that the levels of STX3 protein were down‐regulated in hcmv‐miR‐US33‐5p‐overexpressing cells. Experiments with STX3‐specific siRNA, or with an inhibitor of hcmv‐miR‐US33‐5p confirmed that hcmv‐miR‐US33‐5p‐mediated inhibition of HCMV DNA synthesis and of viral replication are specifically mediated by down‐regulation of STX3 expression.