Heterosis had been increasingly applied in crop production for nearly a century, but the molecular basis of it was poorly understood especially in foxtail millet (Setaria italica). To gain a better understanding of the molecular basis of S.italica heterosis, the isobaric tags for relative and absolute quantification (iTRAQ) based quantitative proteomic analyses was performed between S.italica hybrid Zhangzagu10 and it parents (male parent Zhangzagu10fu and female parent A2). We hope that this study can provide valuable information for the study on the molecular mechanisms of heterosis in hybrid millet.