While Individuals with chronic ankle instability (CAI) exhibit altered ankle joint movement and moments during stance phase of gait, the interaction or dynamic joint stiffness (DJS) between these is not fully understood. Little attention has been placed on DJS during gait, limiting our understanding of how the most common dynamic task during daily life could affect cartilage loading. Do Individuals with CAI exhibit altered ankle DJS and mechanical energy exerted at the ankle joint during stance phase of gait? Eighty-four physically active individuals, consisting of 42 individuals with CAI (12 M and 30 F) and 42 control (12 M and 30 F) participants were recruited in this study. Three-dimensional gait analysis was conducted. The sagittal ankle joint angle and moment during stance phase of walking gait were obtained. Stance phase was divided into three sub-phases: controlled plantarflexion, controlled dorsiflexion, and powered plantarflexion. Ankle DJS was represented by the slope of the joint moment plotted as a function of the joint angle. The coefficient of determination was calculated to determine how accurately data fit a linear model. Net work was calculated by the difference between work produced and absorbed. Further, sex specific exploratory analyses of DJS and work between individuals with and without CAI were conducted. Lower DJS during the controlled plantarflexion (CPF) sub-phase, work produced, and net work was found in the CAI group. Males with CAI exhibited lower ankle moment changes during controlled dorsiflexion (CDF) sub-phase and work absorbed. Females with CAI exhibited lower ankle moment changes during CPF and CDF sub-phases, lower DJS during the CPF sub-phase, and lower net work. Individuals with CAI have alterations in DJS and work relative to uninjured controls. Females with CAI showed greater DJS related alterations, relative to controls, than their male CAI counterparts.