Berberine exhibits polytrophic medicinal roles in various diseases and is safe and effective. However, its role and the underlying mechanism in the replication of herpes simplex virus 1 (HSV-1) remain unreported. This research aimed to determine the functional mechanisms of berberine on HSV-1 infection. We determined the CC50 ( 405.11 ± 15.67  μM) and IC50 ( 45.6 ± 6.84  μM) of berberine on HEK293T cells infected with HSV-1. Berberine inhibited the transcription and translation of HSV-1 activity-related genes (gD, ICP-4, ICP-5, and ICP-8) in HSV-1-infected HEK293T cells dose-dependently. Berberine also inhibited the phosphorylation of MAPK proteins (JNK and p38) and inflammatory responses induced by HSV-1 infection in HEK293T cells dose-dependently. In conclusion, berberine attenuates HSV-1 replication through its activity, infective ability, and inflammatory response. Our research indicated that berberine may be a candidate drug for HSV-1 infection.