Background: Many injury prevention and rehabilitation programs aim to train hamstring and quadriceps co-activation to constrain excessive anterior tibial translation and protect the anterior cruciate ligament (ACL) from injury. However, despite strong clinical belief in its efficacy, primary evidence supporting training co-activation of the hamstrings and quadriceps muscles for ACL injury prevention and rehabilitation is quite limited. Therefore, the purpose of the study presented in this paper was to determine if hamstring-quadriceps co-activation alters knee joint kinematics, and also establish if it affects ACL elongation. Methods: A computed tomography (CT) scan from each participant's dominant leg was acquired prior to performing two step-ups under fluoroscopy: one with 'natural' hamstring-quadriceps co-activation, one with deliberate co-activation. Electromyography was used to confirm increased motor unit recruitment. The CT scan was registered to fluoroscopy for 4-D modeling, and knee joint kinematics subsequently measured. Anterior cruciate ligament attachments were mapped to the 4-D models and its length was assumed from the distance between attachments. Anterior cruciate ligament elongation was derived from the change in distance between those points as they moved relative to each other. Results: Reduced ACL elongation as well as knee joint rotation, abduction, translation, and distraction was observed for the step up with increased co-activation. A relationship was shown to exist for change in ACL length with knee abduction (r = 0.91; p = 0.001), with distraction (r = -0.70; p = 0.02 for relationship with compression), and with anterior tibial translation (r = 0.52; p = 0.01). However, ACL elongation was not associated with internal rotation or medial translation. Medial hamstring-quadriceps co-activation was associated with a shorter ACL (r = -0.71; p = 0.01), and lateral hamstring-quadriceps co-activation was related to ACL elongation (r = 0.46; p = 0.05). Conclusion: Net co-activation of the hamstrings and quadriceps muscles will likely reduce ACL elongation provided that the proportion of medial hamstring-quadriceps co-activation exceeds lateral.