DNA damage repair (DDR) is essential for cancer cell survival and treatment resistance, making it a critical target for tumor therapy. The eukaryotic AAA+ adenosine triphosphatase valosin-containing protein (VCP), which is transported from the cytoplasm into the nucleus, plays a critical role in the DDR process. However, the nuclear translocation and molecular mechanism of VCP for DDR remain elusive. Here, we define VCP as a KPNB1 interacting protein through a combination of chemical and immunoprecipitation mass spectrometry approaches. Further biochemical studies elucidate that KPNB1 directly transports VCP into the nucleus. We also identify withaferin A (WA) as a small molecule that can retard VCP nuclear localization via covalent binding to CYS 158 of KPNB1. Further studies verify WA as an effective antitumor drug candidate via blocking VCP nuclear localization to impact on the DDR pathway in vivo. Our findings underly the unclear VCP's role in DDR in a KPNB1-dependent manner and provide an important theoretical basis for developing small-molecule inhibitors targeting this process.