Artificial parthenogenetic induction via thermal stimuli in silkworm is an important technique that has been used in sericultural production. However, the molecular mechanism underlying it remains largely unknown. We have created a fully parthenogenetic line (PL) with more than 85% occurrence and 80% hatching rate via hot water treatment and genetic selection, while the parent amphigenetic line (AL) has less than 30% pigmentation rate and less than 1% hatching rate when undergoing the same treatment. Here, isobaric tags for relative and absolute quantitation (iTRAQ)-based analysis were used to investigate the key proteins and pathways associated with silkworm parthenogenesis. We uncovered the unique proteomic features of unfertilized eggs in PL. In total, 274 increased abundance proteins and 211 decreased abundance proteins were identified relative to AL before thermal induction. Function analysis displayed an increased level of translation and metabolism in PL. After thermal induction, 97 increased abundance proteins and 187 decreased abundance proteins were identified. An increase in stress response-related proteins and decrease in energy metabolism suggested that PL has a more effective response to buffer the thermal stress than AL. Cell cycle-related proteins, including histones, and spindle-related proteins were decreased in PL, indicating an important role of this decrease in the process of ameiotic parthenogenesis.