Keratinocyte differentiation factor 1 (KDF1) was recently identified as a rare candidate gene of human congenital tooth agenesis. Extensive studies using mouse models have revealed the role of KDF1 in the development of epithelium-derived organs. For example, a study reported that KDF1 mutant mice Kdf1GT/GT exhibited enhanced proliferation and restricted differentiation of epidermal keratinocytes. Furthermore, preliminary findings reported that Kdf1 was mainly expressed in the inner enamel epithelium, which is the pre-differentiated form of ameloblasts, a dental epithelial cell, during tooth development. Thus, we hypothesized that KDF1 plays a crucial role in the differentiation of ameloblast and conducted in vitro experiments to investigate the role of KDF1 in ameloblast differentiation. As the robust evidence to clarify the mechanism through which KDF1 regulates ameloblast differentiation, LS-MS was employed to identify the possible proteins that can interact with KDF1. Thus, the raw data of LS-MS of this project are deposited in iProX database for further investigations. This study is the first to reveal the KDF1-mediated protein interaction network in ameloblast differentiation, not only providing help to broaden the functional cognition of KDF1, but also contributing to enhance the understanding of the gene regulatory network of enamel development.