Spinneret, which locates at the end of the silk gland, is an important tissue for silk spinning. Although it has been discovered for hundreds of years, its particular roles in silk spinning and fiber formation are still unclear. Here we report the first proteome profile of silkworm spinnerets by LC-MS/MS. Totally, 1572 non-redundant proteins and 232 differential expressed proteins were identified in the spinneret from the spinning larvae and the third day of fifth instar larvae. Silk fiber formation related proteins, such as cuticle proteins, ion-transporting proteins, muscular proteins, and energy metabolic proteins, were abundant in the spinneret. By analyzing the signal pathways, we discovered that the processes associated with energy metabolism were active in spinneret. GO enrichment analysis revealed that the differential expressed proteins were involved in energy metabolism, chitin binding, and cuticle construction. The active energy metabolism might provide abundant energy for the muscle contraction, ion and water transporting. The chitin binding and cuticle construction process might provide sufficient shear forces for silk formation. This dataset suggests that the spinneret provided a suitable physiological and biochemical environment for silk formation, and will be helpful for elucidating the functions of spinneret.