In the present study, an image correlation method was used to determine the site-dependent strain in the porcine anterior cruciate ligament (ACL). In particular, the strain of the ACL in the femoral and tibial attachment areas was quantified when the eight knees were subjected to a maximum of 50 N anterior tibial load that was applied using a 6-DOF robotic system. The ACL strains in the anterior, central, and posterior bundles of the medial, middle and lateral layers were determined as a function of the applied anterior load. In addition, the surface of the ACL was observed using a light microscope under no loading condition. Results revealed that the strain in the medial layer of the ACL increased gradually and almost linearly with the increase of anterior load in the midsubstance area. In contrast, the strain in the femoral and tibial attachment areas of the medial layer of the ACL increased rapidly at the beginning of anterior loading and gradually thereafter with significant differences in slope of strain-anterior load curve found in the anterior and posterior bundles. The largest strain in response to 50 N of anterior force was found in the tibial attachment area in medial and middle layers, while the maximum strain was found in the femoral attachment area in the lateral layer. Microscopic observation indicated that crimp structure was more clearly observed in the femoral and tibial attachment areas than in the mid-substance. These microstructural features of the ACL were may be attributable to the higher and load-dependent, nonlinear strain observed in the attachment areas. Our study suggested that the strain in the ACL at full extension is site-and load-dependent in a non-linear manner at the femoral and tibial attachment areas.