Objective：To investigate the effect of a small molecule deubiquitinating enzyme inhibitor，PR-619 on Herpes simplex virus(HSV) replication. Methods：HSV replication and viral protein expression were detected via In-cell Western and Western blot. Viral gene expression on mRNA level was evaluated using Real-time PCR. We studied the inhibitory effect of PR-619 on viral replication，including viral mRNAs transcription and proteins expression. Results：PR-619 down-regulated HSV immediate-early and late gene expression(ICPO，ICP4 and gD) to inhibit viral replication effectively； PR-619 increased amount of ubiquitin-conjugates to suppress intracellular ubiquitin cycle，which may cause inhibitory effect on viral replication.and block viral induced NF-κB pathway activation. Conclusion：HSV effective replication is dependent on functional ubiquitin. Deubiquitinating enzymes is vital for intracellular ubiquitin cycle，which is associated to viral replication. This study could give deep insight into finding novel anti-HSV agents or genital microbicides.