The knowledge of muscle activation patterns when doing a certain task in subjects with anterior cruciate ligament deficiency could help to improve their rehabilitation treatment. The goal of this study is to identify differences in such patterns between anterior cruciate ligament–deficient and healthy subjects during walking. Methods Electromyographic data for eight muscles were measured in a sample of eighteen subjects with anterior cruciate ligament deficiency, in both injured (ipsilateral group) and non-injured (contralateral group) legs, and a sample of ten healthy subjects (control group). The analysis was carried out at two levels: activation-–deactivation patterns and muscle synergies. Muscle synergy components were calculated using a non-negative matrix factorization algorithm. Findings The results showed that there was a higher co-contraction in injured than in healthy subjects. Although all muscles were activated similarly since all subjects developed the same task (walking), some differences could be observed among the analyzed groups. Interpretation The observed differences in the synergy components of injured subjects suggested that those individuals alter muscle activation patterns to stabilize the knee joint. This analysis could provide valuable information for the physiotherapist to identify alterations in muscle activation patterns during the follow-up of the subject’s rehabilitation.