Herpes simplex virus type 1 (HSV-1) infections are one of the most common diseases in human population. HSV-1 causes subclinical, mild to severe diseases, especially in immunocompromised patients. Acyclovir has been used to reduce manifestations of HSV-1 infections. The extensive use of this drug has led to the development of resistant strains. Thus, designing a novel anti-herpes drug with different mechanisms of action is urgently needed. Cellular microRNAs (miRNAs) have direct antiviral effects in addition to their regulatory functions. In this study we used a novel miRNA (hsa-miR-7704), expressed in macrophages, to inhibit HSV-1 lytic infection in HeLa cells. Synthesized hsa-miR-7704 mimics were transfected into HSV-1 infected HeLa cell. The inhibitory effects of the miRNA were evaluated by plaque assay, real time polymerase chain reaction and the viral titers were measured by the 50% tissue culture infective dose (TCID50). The viral titer and cell cytopathic effect were dramatically decreased in HeLa cells transfected with hsa-miR-7704 (50 and 100 nM), compared with HSV-1 infected cells alone or transfected with the mock miRNA control. These results suggest that hsa-miR-7704 inhibits HSV-1 replication efficiently in vitro. This may provide an alternative mechanism to prevent HSV-1 infections.