In this study, a quantitative proteomics of iTRAQ was conducted for rice seeds before and after 24-month natural storage on two hybrid rice cultivars, II You 998 with low storability and the control Boyou 998. More than 5 210 proteins were identified, of which 100 differentially expressed protein (DEP) were significant different. Interestingly, the amount of differentially expressed proteins in the storage period of the seeds with poor storability was significantly higher than that of the control seeds. Furthermore, ribosome pathway was determined to be the main metabolic pathway involved in regulation of seed storability by pathway significant enrichment. Our data showed that more ribosomal proteins were synthesized in the seeds which were not resistant to storage, suggesting that protein biosynthesis might be related to the storage tolerance of seeds. And, the germination percentages of hybrid rice seeds treated with different exogenous protein synthesis inhibitors during the filling stage significantly varied after 2 years storage under natural conditions. Our study showed that treatment of agro-streptomycin, during the filling stage could effectively enhance rice seed storability under natural storage conditions.