Pseudorabies virus (PRV) has spread widely in swine herds since it was introduced into China, and PRV variants have appeared in many regions in China since 2011. Owing to the ongoing emergence of viral mutations, the protective efficacy of PRV vaccines faces great challenges. Gossypol is a polyphenolic natural compound purified from cotton roots, stems, or seeds. This study found that gossypol has a notable inhibitory effect on PRV. Based on the biological effects of gossypol, the anti-PRV effect of gossypol was studied from the aspects of mitochondrial damage, reactive oxygen species, energy metabolism, and autophagy. The results showed that gossypol had no direct inactivation effect on PRV in solution within concentrations of ≤3 M; gossypol did not stimulate interferon- to exert an anti-PRV effect by triggering mitochondrial DNA leakage; the anti-PRV effect of gossypol was reactive oxygen species and ATP-independent in cells; and the PRV-inhibitory effect of gossypol was independent of its induced autophagy. By adding gossypol to PRV-infected cells at different stages of infection and performing virus titer detection, atomic force microscopy, qPCR, fluorescence microscopy, and firefly luciferase activity assays, we found that gossypol exerted an anti-PRV effect by inhibiting the adsorption of PRV on the cell surface.