The natural products piperlongumine (<b>1</b>) and ligustrazine (<b>2</b>) have been reported to exert antiproliferative effects against various types of cancer cells by up-regulating the level of reactive oxidative species (ROS). However, the moderate activities of <b>1</b> and <b>2</b> limit their application. To improve their potential antitumor activity, novel piperlongumine/ligustrazine derivatives were designed and prepared, and their potential pharmacological effects were determined in vitro and in vivo. Among the derivatives obtained, <b>11</b> exerted more prominent inhibitory activities against proliferation of drug-sensitive/-resistant cancer cells with lower IC<sub>50</sub> values than <b>1</b>. Particularly, the IC<sub>50</sub> value of <b>11</b> against drug-resistant Bel-7402/5-FU cells was 0.9 μM, which was about 9-fold better than that of <b>1</b> (IC<sub>50</sub> value of 8.4 μM). Mechanistic studies showed that <b>11</b> demonstrated thioredoxin reductase (TrxR) inhibitory activity, increase of ROS levels, decrease of mitochondrial transmembrane potential levels, and occurrence of DNA damage and autophagy, in a dose-dependent manner, via regulation of DNA damage protein H2AX and autophagy-associated proteins LC3, beclin-1, and p62 in drug-resistant Bel-7402/5-FU cells. Finally, compound <b>11</b> at 5 mg/kg displayed potent antitumor activity in vivo with tumor suppression of 76% (w/w). Taken together, compound <b>11</b> may represent a promising candidate drug for the chemotherapy of drug-resistant hepatocellular carcinoma and warrant more intensive study.