OBJECTIVE To characterize and compare dynamic joint stiffness (DJS) of the ankle in the sagittal plane during natural cadence walking in both genders. METHODS Observation, cross-sectional and matched pairs. METHODS Twenty-one males (mean age=27+/-4.2 years) and 18 females (mean age=22.9+/-4.1 years). METHODS Laboratory at the University de Trás-os-Montes and Alto Douro. The DJS of the ankle was assessed while participants performed barefoot walking at a natural cadence, was measured using a force platform (1000 Hz), and synchronized with a high-speed camera (200 Hz). METHODS DJS of the ankle in males and females was calculated in two sub-phases (the second and the fourth sub-phases) of the stance phase. The Wilcoxon non-parametric matched-pairs test and the Mann-Whitney U non-parametric test were used to analyse difference of DJS of the ankle within and between the groups with p0.05 regarded as significant. RESULTS Male and female participants demonstrated significantly (p<0.005) less DJS during the second sub-phase. DJS was not significantly (p>0.063) different between females (0.0511 N m/kg/ degrees ) and males (0.0596 N m/kg/ degrees ) during the second sub-phase. DJS was significantly (p<0.001) higher in males (0.0844 N m/kg/ degrees ) than females (0.0691 N m/kg/ degrees ) during the fourth sub-phase. CONCLUSIONS Gender-specific DJS characteristics demonstrated by females in this study may be used in preventative training programs designed to promote a greater ability to use contractile components to produce mechanical energy through the ankle.