Tooth is one of highly mineralized loadbearing biocomposite, and its superior strength and resilience arise from a hierarchical and grade organisation of mineral and organic phases [1]. Highly mineralised enamel provides hardness and wear resistance, while collagen-rich dentin contributes toughness and energy dissipation, with the enamel-dentin junction (EDJ) bridges the two layers enabling load transfer and crack arrest [2]. Ageing affects tooth organisation through dentin sclerosis (increased mineralisation and altered mineral-collagen coupling), accumulation of enamel microcracks and wear damage, and reduced compliance at EDJ, collectively modifying the stiffness gradient, crack initiation threshold, and energy dissipation mechanism and resulting in changes of failure modes [3]. These impose significant challenges for repair materials and regeneration strategies of ageing teeth, as effective restoration requires replication of the natural hierarchical organisation and functional gradients to ensure effective integration with aged dental tissues, recovery of load-bearing capacity, and reduced risk of mechanical failure, rather than merely restoring morphology or mineral content. A critical step forward is therefore to establish a mechanistic understanding of age-induced changes in tissue organisation and resultant mechanical behaviours, as the structural and mechanical constraints for effective dental material design and regenerative strategies. In this study, we investigate the structure–function relationships in wild-type, naturally aged (24 months old), and transgenic pre-mature ageing (Klotho-deficient) teeth [4-5] using a comprehensive multiscale characterisation approach. Our approach combines micro-computed tomography (µ-CT), polarised second harmonic generation (pSHG), scanning electron microscopy (SEM), quantitative backscattered electron imaging (qBEI), nanoindentation and tribology, and finite element simulation. Our results reveal pronounced alterations in structural gradients and collagen-mineral organisation, leading to modified deformation behaviours and failure responses. This work provides new insights into bioinspired design of multifunctional graded materials for dental repair and regeneration.