Abnormal joint kinematics are commonly reported in the acute and chronic stages of recovery after anterior cruciate ligament (ACL) injury and have long been mechanistically implicated as a primary driver in the development of posttraumatic osteoarthritis (PTOA). Though strongly theorized, it is unclear to what extent biomechanical adaptations after ACL injury culminate in the development of PTOA, as data that directly connects these factors does not exist. Using a preclinical, noninvasive ACL injury rodent model, our objective was to explore the direct effect of an isolated ACL injury on joint kinematics and the pathogenetic mechanisms involved in the development of PTOA. A total of 32, 16-week-old Long-Evans rats were exposed to a noninvasive ACL injury. Marker-less deep learning software (DeepLabCut) was used to track animal movement for sagittal-plane kinematic analyses and micro computed tomography was used to evaluate subchondral bone architecture at days 7, 14, 28, and 56 following injury. There was a significant decrease in peak knee flexion during walking (p < .05), which had a moderate-to-strong negative correlation (r = −.59 to −.71;​ p < .001) with subchondral bone plate porosity in all load bearing regions of the femur and tibia. Additional comprehensive analyses of knee flexion profiles revealed dramatic alterations throughout the step cycle. This occurred alongside considerable loss of epiphyseal trabecular bone and substantial changes in anatomical orientation. Knee flexion angle and subchondral bone microarchitecture are severely impacted after ACL injury. Reductions in peak knee flexion angle after ACL injury are directly associated with subchondral bone plate remodeling.