Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here, we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knocking down the endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly upon the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and GST pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We further showed that NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taken together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity.IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, few host factors including interferon-stimulated genes (ISGs) are characterized against CSFV. Using a mini-library of porcine ISGs, we identify the porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for the GTPase activity, is essential for antiviral roles of GBP1 against CSFV replication, and binding of the NS5A protein with GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting the host factors and may provide a new strategy for the control of CSF.