The goal of this study was to evaluate muscle activation onset times (MAOT) of both legs during a transition task from double-leg stance (DLS) to single-leg stance (SLS) in anterior cruciate ligament injured (ACLI) (n = 15) and non-injured control subjects (n = 15) with eyes open and eyes closed. Significantly delayed MAOT were found in the ACLI group compared to the control group for vastus lateralis, vastus medialis obliquus, hamstrings medial, hamstrings lateral, tibialis anterior, peroneus longus and gastrocnemius in both vision conditions, for gluteus maximus and gluteus medius with eyes open and for tensor fascia latae with eyes closed. Within the ACLI group, delayed MAOT of tibialis anterior with eyes open and gastrocnemius with eyes closed were found in the injured leg compared to the non-injured leg. All other muscles were not significantly different between legs. In conclusion, the ACLI group showed delayed MAOT not only around the knee, but also at the hip and ankle muscles compared to the non-injured control group. No differences between both legs of the ACLI group were found, except for tibialis anterior and gastrocnemius. These findings indirectly support including central nervous system re-education training to target the underlying mechanisms of these altered MAOT after ACL injury.