To reveal overwintering dormancy (diapause) mechanisms of Culex pipiens pallens (L.), global protein expression differences at three separate time points represent nondiapause, diapause preparation and overwintering diapause phases of Cx. pipiens pallens were compared using iTRAQ. Cx. pipiens pallens females accumulate more lipid droplets during diapause preparation and overwintering diapause maintenance than during the nondiapause phase. A total of 1030 proteins were identified, among which 1020 were quantified and compared. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), Domain and Clusters of Orthologous Groups (COG) analyses revealed key groups of proteins, pathways and domains differentially regulated during diapause preparation and overwintering diapause maintenance phases in this mosquito, including major shifts in energy production and conversion, fatty acid metabolism, the citrate (TCA) cycle, and the cytoskeletal reorganization pathway. Our results provide novel insight into the molecular bases of diapause in mosquitoes and corroborate previously reported diapause-associated features in invertebrates. More interestingly, the phototransduction pathway exists in Cx. pipiens pallens, in particular, actin, rather than other proteins, appears to have substantial role in diapause regulation. In addition, the differential changes in calmodulin protein expression in each stage implicate its important regulatory role of the Cx. pipiens pallens biological clock. Finally, 24 proteins were selected for verification of differential expression using a parallel reaction monitoring strategy. The findings of this study provide a unique opportunity to explore the molecular modifications underlying diapause in mosquitoes and might therefore enable the future design and development of novel genetic tools for improving management strategies in mosquitoes.