The purpose of this study was to compare muscle activity amplitudes and co-contraction in those with anterior cruciate ligament (ACL) reconstruction to healthy controls during stair negotiation. Eighteen participants with unilateral ACL reconstruction and 17 healthy controls performed stair ascent and descent while surface electromyography was recorded from knee and hip musculature. During stair ascent, the ACL group displayed higher gluteus maximus activity (1–50% stance, p = 0.02), higher vastus lateralis:biceps femoris co-contraction (51–100% stance, p = 0.01), and higher vastus lateralis:vastus medialis co-contraction (51–100% stance, p = 0.05). During stair descent, the ACL group demonstrated higher gluteus maximus activity (1–50% stance, p = 0.01; 51–100% stance, p < 0.01), lower rectus femoris activity (1–50% stance, p = 0.04), higher semimembranosus activity (1–50% stance, p = 0.01), higher gluteus medius activity (51–100% stance, p = 0.01), and higher vastus medialis:semimembranosus co-contraction (1–50% stance, p = 0.02). While the altered muscle activity strategies observed in the ACL group may act to increase joint stability, these strategies may alter joint loading and contribute to post-traumatic knee osteoarthritis often observed in this population. Our results warrant further investigation to determine the longterm effects of altered muscle activity on the knee joint following ACL reconstruction.