In the current study, a series of novel quinolinedione-linked sulfonylpiperazine derivatives have been reported as NQO1-directed antitumor agents. A majority of compounds in this study were found to be more effective in resisting the proliferation of cancer cells than that of the positive control 5-Fu and TSA. Among the tested compounds, the derivative 22r exhibited considerable effect (IC, 3.29-5.19 µM) against the proliferation of three NQO1-rich cancer cells (HepG2, MCF-7, and A549), and was recognized to be an excellent NQO1 substrate as revealed by in vitro enzyme reduction assay and molecular docking study with NQO1. In studies on the mechanisms involved, 22r induced reactive oxygen species (ROS) production, caused DNA damage, and induced apoptosis in HepG2 cells. Remarkably, compound 22r exhibited excellent anticancer activity against HepG2 xenograft models in vivo. The study demonstrated that compound 22r provided a promising strategy for the management of malignant tumors.