During seed maturation, the water content of seeds decreases remarkably. Mature seeds can germinate after imbibition since the embryos are protected by mechanism of desiccation tolerance. To better understand the mechanism of desiccation tolerance in seeds, we analyzed the fluctuation of stress-related proteins in the desiccation phase of rice seeds by a real-time RT-PCR and gel-based proteomic approach. Based on the changes in water content of developing rice seeds, we defined stages from the beginning of dehydration (10 to 20 days after flowering) and the desiccation phase (20 to 40 days after flowering). The proteomic analysis revealed that late embryogenesis abundant proteins, small heat shock proteins and antioxidative proteins accumulate at the beginning of dehydration and remain at a high level in the desiccation phase, suggesting that these proteins are involved in acquisition of desiccation tolerance. The fluctuation in levels of mRNA encoding some stress-related proteins did not precisely reflect the change in levels of these proteins. Therefore, proteomic analysis, which provides an accurate assessment of changes in protein levels, is a more efficient technique than transcriptomics for inferring the role of stress-related proteins in rice seeds.