MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression. It has been reported that miRNAs are involved in host-virus interaction, but evidence that cellular miRNAs promote virus replication has been limited. Here, we found that miR-23a promoted the replication of human herpes simplex virus type 1 (HSV-1) in HeLa cells, as demonstrated by a plaque-formation assay and quantitative real-time PCR. Furthermore, interferon regulatory factor 1 (IRF1), an innate antiviral molecule, is targeted by miR-23a to facilitate viral replication. MiR-23a binds to the 3'UTR of IRF1 and down-regulates its expression. Suppression of IRF1 expression reduced RSAD2 gene expression, augmenting HSV-1 replication. Ectopic expression of IRF1 abrogated the promotion of HSV-1 replication induced by miR-23a. Notably, IRF1 contributes to innate antiviral immunity by binding to IRF-response elements to regulate the expression of interferon-stimulated genes (ISGs) and apoptosis, revealing a complex interaction between miR-23a and HSV-1. MiR-23a thus contributes to HSV-1 replication through the regulation of the IRF1-mediated antiviral signal pathway, which suggests that miR-23a may represent a promising target for antiviral treatments.