The aim of this study was to determine the muscle co-activations and joint stiffnesses around the hip, knee, and ankle during different walking speeds and to define the relationships between muscle co-activation and joint stiffness. Twenty-seven healthy subjects (age:​ 19.6 ± 2.2 years, height:​ 176.0 ± 6.0 cm, mass:​ 69.7 ± 8.9 kg) were recruited. Muscle co-activations (CoI) and lower limb joints stiffnesses were investigated during stance phase at different walking speeds using Repeated Measures ANOVA with Sidak post-hoc tests. Correlations between muscle co-activations, joints stiffnesses, and walking speeds were also investigated using Pearson Product Moment correlations. The results indicated that the hip and ankle joints stiffness increased with walking speed (p <​ 0.001) during the weight acceptance phase, and positive correlations were seen between walking speed and Rectus Femoris (RF) and Biceps Femoris (BF) CoI (p <​ 0.001), and a negative correlation was seen between walking speed and tibialis anterior (TA) and lateral gastrocnemius (LG) CoI (p <​ 0.001) during the weight acceptance phase, and the RF/BF CoI during pre-swing. These results provide new information on the variations in muscle co-activation around the hip, knee and ankle joints and their association with joint stiffness, and on the responses of stiffness and muscle co-activation to walking speed. The techniques presented could have further application and help our understanding of the effects of gait retraining and injury mechanisms.