While the soluble proteins of human teeth consist of various extracellular matrix and bioactive proteins, they have not yet been characterized fully. Moreover, the role they play in tooth regeneration is not clear. Analysis of the soluble proteins in human teeth by liquid chromatography-mass spectrometry revealed 147 different ethylenediaminetetraacetic acid-soluble tooth proteins (ESTPs). Of these, 29 had not been shown previously to be present in human teeth. To determine their effect on the in vitro responses of dental pulp stem cells (DPSCs), DPSCs were cultured in ESTP-coated culture plates and three-dimensional scaffolds. The ESTPs significantly enhanced DPSC odontoblast differentiation and mineralization in vitro, but had only partial effect on bone marrow stem cells or adipose tissue stem cells. To test the effect of ESTPs on in vivo dentin and tooth formation, mouse embryonic tooth-forming primordia and xenogenic murine apical bud epithelium/human DPSC composites were treated with ESTPs before implantation under the renal capsule of ICR mice. ESTP treatment promoted the formation of morphologically normal teeth by the tooth-forming primordium regions and enhanced the development of a regular and large dentin structure by the composites. These observations suggest that human ESTPs contain dentinogenic proteins and can promote dentin and tooth formation.