The level of hamstring antagonist activation is thought to be related to knee functionality following anterior cruciate ligament (ACL) injury/surgery as pronounced co-activation can control anterior tibial translation (ATT). The purpose of this study was to examine relationships between knee functionality and hamstring antagonist activation during isokinetic knee extension in ACL deficient (ACLD) and ACL reconstructed (ACLR) patients. Knee functionality was rated using the Cincinnati Knee Rating System for the involved limb of 10 chronic, functional ACLD patients and 27 ACLR patients (14 using a patella tendon (PT) graft and 13 using a semitendinosus/gracilis tendon (STGT) graft). Each subject also performed maximal effort isokinetic knee extension and flexion at 180°. s-1 for the involved limb with electromyographic (EMG) electrodes attached to the semitendinosus (ST) and biceps femoris (BF) muscles. Antagonist activity of the ST and BF muscles was calculated in 10° intervals between 80-10° knee flexion. For the ACLD group, Pearson product moment correlations revealed significant (p<0.05) moderate, positive relationships between knee functionality and ST and BF antagonist activity across the majority of the knee flexion intervals. For both ACLR groups, several significant (p<0.05) moderate, negative associations were found between ST and BF antagonist activity and knee functionality. Amplified hamstring antagonist activity in ACLD patients at flexion angles representative of those at footstrike and deceleration improves knee functionality as increased crossbridge formation increases hamstring stiffness and decreases ATT. Lower-level hamstring activation is sufficient to unload the ACL graft and improve knee functionality in ACLR patients.