Tooth is a good model for studying stem cell driving epithelial and mesenchymal cells development and differentiation. Identifying key factors and pathways governing tooth development will help elucidate the physiological and pathological processes including genetic defects. To investigate the time-dependent proteomic changes of tooth development, label-free quantitative proteomics were conducted for human deciduous molars collected from post conception week (PCW) 12, 15 and 18, encompassing cap to early bell stage. The proteomic profiles reveal dynamic changes of driving proteins and canonical signaling pathways governing tooth development. The proteins with different expression patterns were identified and the protein-protein interaction networks were constructed. Besides, a series of proteins that were potential or novel drivers of tooth morphogenesis had been screened. The polarized expression patterns in odontogenic mesenchymal cell and muscle cell development and the bipolar expression pattern in odontogenic epithelial cell development were revealed. Three candidate proteins (GJA1, PAX9 and KRT15) with different expression patterns were validated by immunohistochemistry. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating tooth development.