Abstract MTH1745, from thermophilic archaea Methanothermobacter thermoautotrophicum, is a protein disulfide isomerase-like protein (PDIL) with a chaperone function and disulfide isomerase activity. Mercuric cations have a high affinity for sulfhydryl groups and consequently inhibit plant growth. Disulfide compounds (e.g., copper–zinc superoxide dismutase, Cu/Zn SOD) and sulfhydryl compounds (e.g., glutathione, phytochelatins, and metallothioneins) play important roles in mercury (Hg) response. To study the relationship between Hg detoxification and PDILs, we overexpressed MTH1745 in Oryza sativa L. cv. Nipponbare by Agrobacterium-mediated transformation. The transgenic rice seedlings displayed Hg tolerance with obvious phenotypes and more effective photosynthesis compared to wild-type plants. Furthermore, lower levels of superoxide anion radicals, hydrogen peroxide, and malondialdehyde were observed in leaves or roots of transgenic plants. Antioxidant enzyme activities of superoxide dismutase and peroxidase were notably higher in transgenic seedlings under different concentrations of mercuric chloride. Moreover, increased content of non-protein thiols, reduced glutathione (GSH), and GSH/GSSG (GSSG, oxidized glutathione) ratio were also observed in the detoxification of Hg. These results indicated that heterologous expression of a PDIL from extremophiles in rice could protect the synthesis, increase stability of proteins, and enhance Hg tolerance in rice.