Technical Abstract: The alfalfa leafcutting bee, Megachile rotundata is used to pollinate various crops in North and South America and Europe. In terms of the number used annually and hectares of crops pollinated M. rotundata is the most intensely managed solitary bee species used in managed pollination programs. A key component in M. rotundata management programs is overwintering storage of the bees. Megachile rotundata overwinters as diapausing prepupae, therefore understanding the physiology of M. rotundata will provide new insights for improving the management of this key pollinator. Currently very little is known about the molecular regulation of insect diapause in general and even less is known about regulation of M. rotundata diapause. In this investigation, Illumina sequencing was employed to examine the physiological transition from diapause to post-diapause development. Illumina identified 1222 differentially regulated genes, 316 of the genes were downregulated and 906 were upregulated in the post-diapausing prepupae. KEGG pathway analysis revealed that the top three associated pathways with the diapause upregulated genes are starch and sucrose metabolism, glycolysis / gluconeogenesis and purine metabolism and for diapause downregulated genes the top three pathways are purine metabolism, pyrimidine metabolism, and phosphatidylinositol signaling system.