Many insects in temperate regions overwinter in diapause. In these insects, one of the metabolic adaptations to cold stress is the synthesis of responsive proteins. Using proteomic analysis, an investigation aimed to a better understanding of the molecular adaptation mechanisms to cold stress was carried out in Ostrinia furnacalis larva. Proteins were extracted from the larval hemolymph collected from both control and overwintering larva. By polyethylene glycol precipitation, approximately 560 protein spots were separated and visualized on two‐dimensional (2D) gels after silver staining. Eighteen protein spots were found to be upregulated in overwinter larval plasma in different patterns. As an initial work, 13 of these proteins were identified using MALDI TOF/TOF MS. The differentially overexpressed proteins include heat shock 70 kDa cognate protein, small heat shock protein (sHSP), putative aliphatic nitrilase, arginine kinase, phosphoglyceromutase, triosephosphateisomerase, and glutathione transferase. Alterations in the levels of these proteins were further confirmed by qPCR. This study is the first analysis of differentially expressed plasma proteins in O. furnacalis diapause larvae under extremely low temperature conditions and gives new insights into the acclimation mechanisms responsive to cold stress. Our results also support the idea that energy metabolism, alanine and proline metabolism, and antioxidative reaction act in the cold acclimation of O. furnacalis diapause larvae.