Individuals with anterior cruciate ligament (ACL) reconstruction often display abnormal gait mechanics reflective of a “stiff-knee” gait (i.e., reduced knee flexion angles and moments). However, dynamic knee stiffness, which is the dynamic relationship between the position of the knee and the moment acting on it, has not been directly examined during walking in individuals with ACL reconstruction. Here, we aimed to evaluate dynamic knee stiffness in the involved compared to the uninvolved limb during weight-acceptance and mid-stance phases of walking. Twenty-six individuals who underwent ACL reconstruction (Age:​ 20.2 ± 5.1 yrs., Time post-op:​ 7.2 ± 0.9 mo.) completed an overground walking assessment using a three-dimensional motion capture system and two force plates. Dynamic knee stiffness (Nm/°) was calculated as the slope of the regression line during weight-acceptance and midstance, obtained by plotting the sagittal plane knee angle versus knee moment. Paired t-tests with Bonferroni corrections were used to compare differences in dynamic stiffness, knee excursions, and moment ranges between limbs during both stance phases. Greater dynamic knee stiffness was found in the involved compared with the uninvolved limb during weight-acceptance and mid-stance (p < 0.01). Knee flexion and extension excursions were reduced in the involved limb during both weight-acceptance and mid-stance, respectively (p < 0.01). Sagittal plane knee moment ranges were not different between limbs during weight-acceptance (p = 0.1);​ however, the involved limb moment range was reduced relative to the uninvolved limb during mid-stance (p < 0.01). These results indicate that individuals with ACL reconstruction walk with a stiffer knee throughout stance, which may influence knee contact forces and could contribute to the high propensity for post-traumatic knee osteoarthritis development in this population.