The enthesis is the attachment site of tendon and ligament to bone. Fibrocartilaginous entheses are located at the epiphysis or the apophysis of the bone. Primordial entheses arise from Scx+/Sox9+ progenitors during embryonic development to form as the junction between tendons/ligaments and hyaline cartilage. Fibrocartilaginous entheses consist of four graded tissue layers including tendon, unmineralized and mineralized fibrocartilage, and subchondral bone with varying degrees of stiffness. The mineral gradient of the enthesis is thought to be particularly important for limiting stress concentrations at the bone-tendon interface. However, it remains uncertain how such fine structure is constructed during postnatal growth. Taking advantage of the cryofilm method reported by Kawamoto, we performed histological and atomic force microscopy analyzes on cryosections of non-decalcified hard tissues to examine formation of the fibrocartilaginous enthesis. Development of mineralized fibrocartilage was followed by the expansion of unmineralized fibrocartilage after the decreased ALP activity in the mineralization front. Calcein labelling revealed that the mineralization front in the enthesis extends unidirectionally towards the midsubstance of the Achilles tendon. We found that sclerostin, which antagonizes canonical Wnt/b-catenin and BMP signaling, is expressed in mature mineralized fibrocartilage adjacent to subchondral bone. In Scx deficient mice with decreased mechanical loading due to defective tendon formation, both fibrocartilage and hyaline cartilage formation was impaired and sclerostin expression was markedly decreased. Loss of the Sost gene, which encodes sclerostin, resulted in increased bone mineral density and higher stiffness in the fibrocartilaginous enthesis. Thus, sclerostin marks mature fibrochondrocytes to modulate the stiffness gradient to maintain tissue integrity of the fibrocartilaginous enthesis.