HMGB1 is a highly conserved nuclear protein with functions that depend on its biological environment, which are linked to structural differences in the protein. Inside the cell, HMGB1 adopts a reduced form, regulating DNA transcription. In contrast, in the extracellular environment, it exists in a form with a closed disulfide bridge within the A-box motif playing a role in inflammation. We analyzed the stability of HMGB1 in these two redox states using differential scanning fluorimetry (nanoDSF), which enables high-precision thermal unfolding measurements with minimal protein quantities — something not previously feasible for HMGB1. The A-box domain was found to unfold reversibly in both redox forms, unlike the B-box. Surprisingly, the reduced form showed lower thermal stability but higher enthalpy of unfolding, indicating that it is enthalpically favorable and suggesting a significant difference in entropy contributions. For full-length HMGB1, both redox variants displayed similar thermal stability. However, only the reduced form was able to refold after unfolding; the disulfide form could not return to its native structure. Additionally, the reduced full-length variant exhibited a decrease in unfolding enthalpy, likely due to the destabilizing effect of its negatively charged C-terminal tail. Overall, the redox state has a strong influence on HMGB1's thermodynamic behavior. These thermodynamic differences can be linked to the protein's dual functionality: enhanced flexibility is beneficial for DNA transcription inside the nucleus. At the same time, increased conformational stability is advantageous for extracellular protein-protein recognition pathways.