Despite advances in our understanding of anterior cruciate ligament (ACL) anatomy and function, the change in position of the ACL during tibial rotation is not well understood. The purpose of this study was to quantify and compare the movements of the anteromedial (AM) and posterolateral (PL) bundles of the ACL during 15° of medial and lateral rotation (with & without a shear force). Cadaveric knees (12 male/12 female) were dissected and mounted at 90° of knee flexion. Anthropometric features of the ACL and distal femur were recorded, and each bundle was marked at: femoral attachment (FA), midpoint of ligament (MP), and tibial attachment (TA). Digital images of ACL motion in the frontal plane were taken as the tibia was rotated about a fixed femur. Using digitizing software, the change in position of the markers was quantified. Measurements suggested the ACL pattern of motion was consistent between sexes, regardless of shear force. The greatest amount of movement of both the AM and PM bundles occurred at the TA marker. The FA marker moved more during medial rotation, and the MP and TA markers moved more with lateral rotation. The 20 lb-shear force affected medial rotation most. This study is the first to quantify movement of the ACL during medial and lateral tibial rotation. Data should assist surgeons to select a graft position that is capable of replicating the rotational movement of the native ACL, and help improve three-dimensional stability of the ACL reconstructed knee. Clin. Anat. 30:861-867, 2017. © 2017 Wiley Periodicals, Inc.