To investigate the effects of H₂S on the depolymerization and hydrolysis of seed globulins, hydrated brassica seeds were exposed to a hydrogen sulfide donor (NaHS) under programmed cooling conditions (–60 ℃/h). The cryotolerance of the seeds and the extent of globulin hydrolysis were assessed. Germination rates were evaluated following programmed cooling. Differential scanning calorimetry (DSC) was employed to determine the onset temperature of ice formation and the associated enthalpy change. Total seed proteins were extracted for two-dimensional electrophoresis (2-DE), and differentially expressed protein spots were identified using mass spectrometry. Small-molecular-weight peptides were isolated, and ice crystal formation patterns were analyzed via cryomicroscopy. Additionally, the concentrations of free thiols and disulfide bonds were quantified. The results demonstrated that the exogenous application of 5 mmol/L NaHS significantly enhanced the germination rate of hydrated brassica seeds under programmed cooling conditions. The onset temperature of internal ice formation was markedly delayed in NaHS-treated seeds, and the total thiol content was substantially higher in the NaHS-treated group compared to the control. Two-dimensional electrophoresis revealed two differentially expressed protein spots in the low-molecular-weight region (<25 kDa). Mass spectrometry identified these spots as 2 S albumin, 12 S globulin, and 40 S ribosomal protein. These smaller peptides exhibited lower ice nucleation temperatures and formed smaller, more rounded ice crystals during cooling. In conclusion, the promotion of disulfide bond reduction and the degradation of globulin-derived small peptides represent critical mechanisms by which NaHS enhances the survival of water-containing brassica seeds under low-temperature stress.