Background and ObjectiveThe structural and functional integrity of bone–periodontal ligament (PDL)–cementum complex stems from the load‐bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone–PDL–cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses.Material and MethodsMultilabeling technique was performed on hemimandibles of 3 wk and 3 mo‐old scleraxis–GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton.ResultsCD146+ cells adjacent to CD31+ vasculature were identified at PDL–bone enthesis. NG2+ cells were located at coronal bone–PDL and apical cementum–PDL entheses in the 3‐wk‐old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL–bone and PDL–cementum entheses. Bone sialoprotein was prominent at the apical region of 3‐wk‐old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk.ConclusionColocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load‐bearing bone–PDL–tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo‐ and cemento‐blastic lineages at the PDL–bone and PDL–cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.