To reveal the difference among three mature Jatropha curcas seeds (JcVH, variant with high lipid content; JcW, wild type and JcVL, variant with low lipid content) with different lipid content, comparative proteomics was employed to profile the changes of oil body (OB) associated protein species by using gels-based proteomic technique. Eighty-three protein species were successfully identified through LTQ-ES-MS/MS from mature JcW seeds purified OBs. Two-dimensional electrophoresis analysis of J. curcas OB associated protein species revealed they had essential interactions with other organelles and demonstrated that oleosin and caleosin were the most abundant OB structural protein species. Twenty-eight OB associated protein species showed significant difference among JcVH, JcW and JcVL according to statistical analysis. Complementary transient expression analysis revealed that calcium ion binding protein (CalBP) and glycine-rich RNA binding protein (GRP) were well targeted in OBs apart from the oleosins. This study demonstrated that ratio of lipid content to caleosins abundance was involved in the regulation of OB size, and the mutant induced by ethylmethylsulfone treatment might be related to the caleosin like protein species. These findings are important for biotechnological improvement with the aim to alter the lipid content in J. curcas seeds. The economic value of Jatropha curcas largely depends on the lipid content in seeds which are mainly stored in the special organelle called oil bodies (OBs). In consideration of the biological importance and applications of J. curcas OB in seeds, it is necessary to further explore the components and functions of J. curcas OBs. Although a previous study concerning the J. curcas OB proteome revealed oleosins were the major OB protein component and additional protein species were similar to those in other oil seed plants, these identified OB associated protein species were corresponding to the protein bands instead of protein spots in the electrophoresis gels. Furthermore, the interaction of OB associated protein species and their contribution to OB formation and stabilization are still blank. In this study, with the overall object of profiling OB protein species from mature J. curcas seeds with different lipid content, we provided a setting of comparative OB proteomics with biochemical data and transient expression to explore the core of OB associated protein species involved in the regulation of OB size and lipid accumulation. The results were important for biotechnological improvement with the aim to a global modification of lipid storage in J. curcas seeds. Meanwhile, this study gave insight into possible associations between OBs and other organelles in mature J. curcas seeds. It may represent new aspects of the biological functions of the OBs during the oil mobilization. Combined the technique of transient transformation, a newly reported protein species, glycine-rich RNA binding protein (GRP) was successfully targeted in OBs. Therefore, further molecular analysis of these protein species is warranted to verify this association and what role they have in OBs.