Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gammaherpesvirus that establishes persistent lifelong infection in host cells. In order to establish successful infection, KSHV has evolved numerous immune evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during primary KSHV infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on several RNA viruses; however, its function in DNA virus infection is less well understood. In this study, we found that KSHV infection increases both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-κB pathway. The overexpression of GBP1 significantly inhibited KSHV infection, while the knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filaments. Finally, we demonstrated that replication and transcription activator (RTA) promotes the degradation of GBP1 through a proteasome pathway. Taken together, these results provide a new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity, and suggest the critical role of RTA in the evasion of the innate immune response during primary infection by KSHV. GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting the nuclear delivery of KSHV virions via the disruption of actin filaments. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome-mediated pathway. Taken together, our results elucidate a novel mechanism of GBP1 anti-KSHV activity and emphasize the critical role of RTA in KSHV evasion of the host immune system during primary infection.