Pseudorabies virus (PRV) accounts for a critical swine disease incurring economic losses worldwide. Several PRV vaccines are commercially available but these vaccines are effective against only certain prevalent PRV strains in China. To prevent PRV-induced latent infection and decrease the pathogenicity, novel anti-PRV drugs are required to prevent PRV infection. Natural products show exceptional structural diversity representing an important source for developing novel therapeutic agents. Quercetin is a flavonoid with anti-oxidant, anti-cancer, anti-bacterial and anti-viral activities. This study involved quercetin for studying the anti-PRV function in vitro and in vivo. Quercetin was found to significantly decrease the PRV virulent strain HNX at a half-maximal inhibitory concentration (IC50) of 2.618 μM and selectivity index 229. This anti-PRV activity of quercetin was found to be dose-dependent. Furthermore, quercetin also inhibited a wide the infections by a spectrum of PRV strains like HNX, Ea, Bartha and Fa strain. These virucidal effects of quercetin suggest the interaction between these molecules and viral particles, and quercetin is responsible for inhibiting the adsorption of PRV infections. The silico assays suggesting that quercetin might interact with the gD-protein on the surface of the PRV important for viral infection. Additional, the quercetin plantar injection protected the mice from the lethal challenge, decreasing the PRV-infected mice's brain viral loads and mortality. These results provides a anti-PRV strategy and contribute to drug discovery and development.