In order to investigate the differential expression of proteins related to pupation in silkworm, the fat body proteins were extracted from larvae of 5th day of 5th instar, un-pupated larvae and pupae of pupating day of multivoltine variety “da zao”. The optimized IPG-IEF/SDS-PAGE (2-DE) was used to analyze the differential expression of proteome in different developmental stage during pupation. The results showed that 66 proteins exhibited a more than two-fold differential expression, in which 43 proteins were down-regulated while 23 proteins were up-regulated. Additionally, 14 proteins with stage-specific expression characteristics were also observed. From 25 proteins with three-fold differential expression, 12 proteins were identified by MALDI-TOF/TOF MS, such as actin, calponin-like protein, NADH, beta-tubulin, receptor for activated protein kinase C, IMP cyclohydrolase, tropomyosin, antichymotrypsin precursor and 30K protein precursor etc. These results suggest that the differentially expressed proteins (enzymes) are related to the regulation of silkworm metamorphosis or sex differentiation, which provides a new experimental basis for better understanding the physiological functions and the mechanism of gene expression in silkworm fat body.