A series of novel β-lapachone analogs was designed and synthesized by replacing pyran ring of β-lapachone with tetrahydropyrimidinethione moiety of monastrol. These hybrids had potent antiproliferative activity against NQO1-rich cell lines (HepG2 and A549), while NQO1-defficient cell lines (H596 and LO2) were less sensitive to these hybrids. Dicoumarol partially inhibited the activity of these compounds against A549 cell lines, indicating that the activation of biological reduction mediated by NQO1 might partly affect the antiproliferative effects. NQO1 assay and docking study demonstrated 4j was a good substrate of NQO1. Furthermore, as suggested by cellular mechanistic research concerning antitumor activity, the representative compound 4j resulted in ROS production depending on NQO1, then oxidative stress triggered apoptotic cell death. Importantly, 4j significantly suppressed cancer growth in HepG2 xenograft models without obvious toxicity, suggesting that 4j deserve further research as potent antitumor agents for cancer therapy.