Pseudorabies virus (PRV), the causative pathogen of Aujeszky's disease, is one of the most important pathogens threatening the global pig industry. Although vaccination has been used to prevent PRV infection, the virus cannot be eliminated in pigs. Thus, novel antiviral agents as complementary to vaccination are urgently needed. Cathelicidins (CATHs) are host defense peptides that play an important role in the host immune response against microbial infections. In the study, we found that the chemical synthesized chicken cathelicidin B1 (CATH-B1) could inhibit PRV regardless of whether CATH-B1 was added pre-, co-, or post-PRV infection and . Furthermore, coincubation of CATH-B1 with PRV directly inactivated virus infection by disrupting the virion structure of PRV and mainly inhibited virus binding and entry. Importantly, pretreatment of CATH-B1 markedly strengthened the host antiviral immunity, as indicated by the increased expression of basal interferon-β (IFN-β) and several IFN-stimulated genes (ISGs). Subsequently, we investigated the signaling pathway responsible for CATH-B1-induced IFN-β production. Our results showed that CATH-B1 induced phosphorylation of interferon regulatory transcription factor 3 (IRF3) and further led to production of IFN-β and reduction of PRV infection. Mechanistic studies revealed that the activation of Toll-like receptor 4 (TLR4), endosome acidification, and the following c-Jun N-terminal kinase (JNK) was responsible for CATH-B1-induced IRF3/IFN-β pathway activation. Collectively, CATH-B1 could markedly inhibit PRV infection via inhibiting virus binding and entry, direct inactivation, and regulating host antiviral response, which provided an important theoretical basis for the development of antimicrobial peptide drugs against PRV infection. Although the antiviral activity of cathelicidins could be explained by direct interfering with the viral infection and regulating host antiviral response, the specific mechanism of cathelicidins regulating host antiviral response and interfering with pseudorabies virus (PRV) infection remains elusive. In this study, we investigated the multiple roles of cathelicidin CATH-B1 against PRV infection. Our study showed that CATH-B1 could suppress the binding and entry stages of PRV infection and direct disrupt PRV virions. Remarkably, CATH-B1 significantly increased basal interferon-β (IFN-β) and IFN-stimulated gene (ISG) expression levels. Furthermore, TLR4/c-Jun N-terminal kinase (JNK) signaling was activated and involved in IRF3/IFN-β activation in response to CATH-B1. In conclusion, we elucidate the mechanisms by which the cathelicidin peptide direct inactivates PRV infection and regulates host antiviral IFN-β signaling.