Injuries to the anterior cruciate ligament (ACL) often occur at the ligament-to-bone insertion site; thus, an in-depth understanding of the native insertion is critical in identifying the etiology of failure and devising optimal treatment protocols for ACL injuries. The objective of this study is to conduct a systematic characterization of the ACL-to-bone interface, focusing on structural and compositional changes as a function of age. Using a bovine model, three age groups were studied: Neonatal (1-7 days old), Immature (2-6 months old), and Mature (2-5 years old). The distribution of types I, II, X collagen, decorin, cartilage oligomeric matrix protein (COMP), glycosaminoglycan (GAG), alkaline phosphatase (ALP) activity, and minerals at the ACL-to-bone insertion were examined. Additionally, cell aspect ratio, size, and distribution across the insertion were quantified. The ACL-to-bone insertion is divided into four regions: ligament, nonmineralized interface, mineralized interface, and bone. Both region-dependent and age-dependent structural and compositional changes at the insertion site were observed in this study. The interface in the skeletally immature group resembled articular cartilage, while the adult interface was similar to fibrocartilaginous tissue. Age-dependent changes in extracellular matrix composition (type X collagen, sulfated glycosaminoglycan), cellularity, ALP activity, and mineral distribution were also found. Marked differences in collagen fiber orientation between the femoral and tibial insertions were observed, and these differences became more pronounced with age.