ObjectiveTo investigate the difference in bilateral lower limb muscle synergy mode during gait in patients after unilateral anterior cruciate ligament reconstruction. MethodsElectromyography from bilateral lower limb muscles during gait were collected from twelve male and eight female patients after unilateral anterior cruciate ligament reconstruction in Affiliated Hospital of Wuhan Sports University, from April to June, 2023. The data were analyzed using non-negative matrix decomposition algorithm to extract the number of muscle synergies in the affected and unaffected legs, the time to peak activation of muscle synergies and the relative weights of the muscles. ResultsSix types of muscle synergy were identified in the unaffected leg of males during gait, while five types were identified in the affected leg, lacking synergy 2 that mainly from the tibialis anterior muscle. Six types of muscle synergy were identified in both legs in females during gait. There was no significant difference in the time to peak activation of muscle synergies between both legs in males (P > 0.05). However, the time to peak activation of muscle synergies increased in females in the affected leg for synergy 3 and synergy 5 (P < 0.05). The relative weight of the rectus femoris was lower in synergy 1 in the affected leg in males (P < 0.05). For female, the relative weight of the vastus lateralis was higher and the relative weight of the biceps femoris was lower in synergy 2 in the affected leg in females (P < 0.05); while the relative weight of the rectus femoris was lower in synergy 3 (P < 0.05), and the relative weight of the biceps femoris was lower in synergy 6 (P < 0.05). ConclusionMales would freeze the muscle synergy dominating ankle dorsiflexion in affected leg to enhance ankle stability, and reduce the relative weight of rectus femoris during the loading response phase to weaken the knee landing cushioning. However, females would delay the activation of synergies dominating in loading response phase and the mid-stance phase, enhance the relative weight of vastus lateralis during the loading response phase, and reduce the relative weights of rectus femoris in the loading response phase and the relative weight of biceps femoris in the mid-stance phase, to limit knee flexion.