The nascent thyroglobulin (Tg) multimer molecule, which is generated during the initial fate of Tg in ER, undergoes the rapid reductive depolymerization. In an attempt to determine the depolymerization process, various types of Tg multimers, which were generated from deoxy­cholate-treated/reduced Tg, partially unfolded Tg or partially unfolded/reduced Tg, were subjected to various GSH (reduced glutathione) reducing systems using protein disulfide isomerase (PDI), glutathione reductase (GR), glutaredoxin or thioredoxin reductase. The Tg multimers generated from deoxycholate-treated/reduced Tg were depolymerized readily by the PDI/GSH system, which is consistent with the reductase activity of PDI. The PDI/GSH-induced depolymerization of the Tg multimers, which were generated from either partially unfolded Tg or partially unfolded/reduced Tg, required the simultaneous inclusion of glutathione reductase, which is capable of reducing glutathionylated mixed disulfide (PSSG). This suggests that PSSG was generated during the Tg multimerization stage or its depolymerization stage. In particular, the thioredoxin/thioredoxin reductase system or glutaredoxin system was also effective in depolymerizing the Tg multimers generated from the unfolded Tg. Overall, under the net GSH condition, the depolymerization of Tg multimers might be mediated by PDI, which is assisted by other reductive enzymes, and the mechanism for depolymerizing the Tg multimers differs according to the type of Tg multimer containing different degrees and types of disulfide linkages.