Pseudorabies virus (PRV), in the family Herpesviridae, is a pathogen of Aujeszky's disease, which causes great economic losses to the pig industry. Recent outbreaks of Pseudorabies imply that new control measures are urgently needed. The present study shows that kaempferol is a candidate drug for controlling PRV infection, as it possesses the ability to inhibit PRV replication in a dose-dependent manner in vitro. Kaempferol at a concentration of 52.40 μmol L–1 could decrease PRV-induced cell death by 90％. With an 50％ inhibitory concentration (IC50) value of 25.57 μmol L–1, kaempferol was more effective than acyclovir (positive control) which has an IC50 value of 54.97 μmol L–1. A mode of action study indicated that kaempferol inhibited viral penetration and replication stages, decreasing viral loads by 4- and 30-fold, respectively. Addition of kaempferol within 16 h post infection (hpi) could significantly inhibit virus replication, and viral genome copies were decreased by almost 15-fold when kaempferol was added at 2 hpi. Kaempferol regulated the NF-κB and MAPKs signaling pathways involved in PRV infection and changed the levels of the target genes of the MAPKs (ATF-2 and c-Jun) and NF-κB (IL-1α, IL-1β and IL-2) signaling pathways. The findings of the current study suggest that kaempferol could be an alternative measure to control PRV infection.