Targeting thioredoxin reductase (TXNRD) with low-weight molecules is emerging as a high-efficacy anti-cancer strategy in chemotherapy. Sanguinarine has been reported to inhibit the activity of TXNRD1, indicating that benzophenanthridine alkaloid is a fascinating chemical entity in the field of TXNRD1 inhibitors. In this study, the inhibition of three benzophenanthridine alkaloids, including chelerythrine, sanguinarine, and nitidine, on recombinant TXNRD1 was investigated, and their anti-cancer mechanisms were revealed using three gastric cancer cell lines. Chelerythrine and sanguinarine are more potent inhibitors of TXNRD1 than nitidine, and the inhibitory effects take place in a dose- and time-dependent manner. Site-directed mutagenesis of TXNRD1 and in vitro inhibition analysis proved that chelerythrine or sanguinarine is primarily bound to the Sec498 residue of the enzyme, but the neighboring Cys497 and remaining N-terminal redox-active cysteines could also be modified after the conjugation of Sec498. With high similarity to sanguinarine, chelerythrine exhibited cytotoxic effects on multiple gastric cancer cell lines and suppressed the proliferation of tumor spheroids derived from NCI-N87 cells. Chelerythrine elevated cellular levels of reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress. Moreover, the ROS induced by chelerythrine could be completely suppressed by the addition of N-acetyl-L-cysteine (NAC), and the same is true for sanguinarine. Notably, Nec-1, an RIPK1 inhibitor, rescued the chelerythrine-induced rapid cell death, indicating that chelerythrine triggers necroptosis in gastric cancer cells. Taken together, this study demonstrates that chelerythrine is a novel inhibitor of TXNRD1 by targeting Sec498 and possessing high anti-tumor properties on multiple gastric cancer cell lines by eliciting necroptosis. © 2023 by the authors.