To determine the relationship between protein expression and insect diapause, a proteomic approach was used to investigate the proteins extracted from larvae of the wheat blossom midge Sitodiplosis mosellana Gehin at different developmental stages, including pre-diapause, over-summering diapause, over-wintering diapause and post-diapause. Using 2-DE gels stained with coomassie brilliant blue, about 300 protein spots were detected in the extracts of pre-diapause larvae and 275 for those in each of the other stages. There were 91, 92 and 95 protein spots that showed more than a 2-fold change in abundance in the over-summering diapause, over-wintering diapause and post-diapause stages compared with pre-diapause. Eight protein spots, which showed the greatest difference in the larvae at different stages of diapause, were analyzed using Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Seven of them were successfully identified from their peptide mass fingerprints using the NCBInr database. They were proopiomelanocortin, NADH dehydrogenase subunit 1 and F10F2.5, which were up-regulated or unique to pre-diapause larvae, IKK interacting protein isoform 2 up-regulated in diapause and post-diapause larvae, GA10647-PA unique in over-wintering diapause larvae, purple CG16784-PB isoform B and B0228.6 up-regulated in over-summering and over-wintering diapause larvae. The potential functions of these proteins during wheat blossom midge diapause are discussed.