Thioredoxin reductase 1 (TXNRD1) has been identified as one of the promising chemotherapeutic targets in cancer cells. Therefore, a novel TXNRD1 inhibitor could accelerate chemotherapy in clinical anticancer research. In this study, glaucocalyxin A (GlauA), a natural diterpene extracted from Rabdosia japonica var. glaucocalyx, was identified as a novel inhibitor of TXNRD1. We found that GlauA effectively inhibited recombinant TXNRD1 and reduced its activity in gastric cancer cells without affecting the enzyme's expression level. Mechanistically, the selenocysteine residue (U498) of TXNRD1 was irreversibly modified by GlauA through a Michael addition. Additionally, GlauA formed a covalent adduct with glutathione (GSH) and disrupted cellular redox balance by depleting cellular GSH. The inhibition of TXNRD1 and depletion of GSH by GlauA conferred its cytotoxic effects in spheroid culture and Transwell assays in AGS cells. The disulfide stress induced cytotoxicity of GlauA could be mitigated by adding reducing agents, such as DTT and β-ME. Furthermore, the FDA-approval drug auranofin, a TXNRD1 inhibitor, triggered oligomerization of the cytoskeletal protein Talin-1 in AGS cells, indicating that inhibiting TXNRD1 triggered disulfide stress. In conclusion, this study uncovered GlauA as an efficient inhibitor of TXNRD1 and demonstrated the potential of TXNRD1 inhibition as an effective anticancer strategy by disrupting redox homeostasis and inducing disulfide stress.