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Objectives The local structures and mechanical properties within tooth dentin-enamel-junction (DEJ) regions have been focused for numerous studies. The reported results, however, remain inconsistent particularly on the functional width and gradient architecture of the DEJ. The current study aims at systematically determining the mechanical gradient of the DEJ at different intratooth locations such that a clearer understanding on the tooth properties and the potential correlations with the tooth function could be obtained. Methods We re-examined how mechanical properties such as elastic modulus and hardness transitioned from those of dentin to those of enamel using combined static and dynamic nanoindentation mapping techniques. A new mapping method and associated image processing procedures were developed to improve the measurement accuracy and resolution. Results A thin, sigmoidally-transitioned interphase layer of the DEJ was identified with an accurate functional width of 2–3 μm. The DEJ width and gradient architecture were found intratooth location-dependent, with the DEJ at the occlusal sites being wider and transitioning smoother than that at the cervical sites. Such different widths and architectures of the interphase layer at sites subjected to different types and magnitudes of loadings during mastication could promote more efficient stress transferring between enamel and dentin without compromising the overall stiffness of the tooth. Significance The presented study not only adds our understanding in the local mechanical properties within tooth DEJ regions, it could also further advance the development of DEJ-mimetic, functional gradient interphase for strong and ultra-durable jointing between dissimilar materials.