This study determined the insertion angle at the porcine anterior cruciate ligament (ACL) enthesis under joint loading to provide information on the structure and mechanical function of the enthesis. Ten intact porcine knee joints were harvested, and an anterior tibial load was applied using a robotic testing system. After dissecting a portion of the ACL enthesis along ligament fibers, the remaining enthesis was imaged using a digital microscope while reproducing the three-dimensional intact knee motion. Fiber orientation angles (FOAs) in the enthesis region (0-300 µm from the ligament-bone boundary) and the ligament region (500-2000 µm from the ligament-bone boundary) were analyzed in the femoral and tibial entheses of the anteromedial bundle (AMB) of the ACL under loading. On the femoral side, the FOA in the enthesis region was significantly higher than that in the ligament region by approximately 10 degrees under loading (n = 5, p <
0.05 in paired t-test). In contrast, the FOAs in the enthesis and ligament regions on the tibial side were nearly equal under loading, with no significant difference (n = 5, p >
0.15 in paired t-test). Histological examination indicated that uncalcified fibrocartilage (UF) was abundant in the enthesis region of the AMB femoral enthesis while the UF was not observed in the enthesis region of the AMB tibial enthesis. Thus, the current data suggest that the regional dependence and independence in FOA are caused by the presence or absence of UF and contributes to a moderate and subtle load-transduction in the ACL enthesis.