Targeting specific molecules is a promising cancer treatment because certain types of cancer cells are dependent on specific oncogenes. This strategy led to the development of therapeutics that use monoclonal antibodies or small-molecule inhibitors. However, the continued development of novel molecular targeting inhibitors is required to target the various oncogenes associated with the diverse types and stages of cancer. Obtusilactone B is a butanolide derivative purified from Machilus thunbergii. In this study, we show that obtusilactone B functions as a small-molecule inhibitor that causes abnormal nuclear envelope dynamics and inhibits growth by suppressing vaccinia-related kinase 1 (VRK1)-mediated phosphorylation of barrier-to-autointegration factor (BAF). BAF is important in maintaining lamin integrity, which is closely associated with diseases that include cancer. Specific binding of obtusilactone B to BAF suppressed VRK1-mediated BAF phosphorylation and the subsequent dissociation of the nuclear envelope from DNA that allows cells to progress through the cell cycle. Obtusilactone B potently induced tumor cell death in vitro, indicating that specific targeting of BAF to block cell cycle progression can be an effective anticancer strategy. Our results demonstrate that targeting a major constituent of the nuclear envelope may be a novel and promising alternative approach to cancer treatment.