Background: The regulation of ankle joint stiffness by combination of activation of plantarflexor and dorsiflexor during gait has not been investigated in patients with hemiparesis. The objective of the present study was to examine the relationship between combination of activation of ankle muscles and quasi-joint stiffness (QJS) during the stance phase of gait. Methods: The activation of the medial head of the gastrocnemius (MG), soleus, and tibialis anterior, gait parameters were collected from 19 patients with hemiparesis due to stroke and from 12 healthy controls using a three-dimensional motion analysis system. The indexes of reciprocal activation and coactivation were calculated from the ratio of plantarflexor to dorsiflexor activation and magnitude of coactivation (MC), which is computed by multiplying an index of simultaneous activation of ankle muscles by plantarflexor activation. Results: QJS was significantly correlated with MC of MG on the paretic side, whereas it correlated with the ratio of MG (r = 0.63, p < 0.05) in healthy controls and the ratio of MG (r = 0.67, p < 0.05) and soleus (r = 0.61, p < 0.05) on the non-paretic side in midstance. Furthermore, QJS on the paretic side was lower than that on the non-paretic side and in healthy controls (p < 0.05). Significance: Our findings support that the regulation of QJS in midstance by reciprocal activation is altered on the paretic side, whereas it may be regulated by reciprocal activation and enhanced by relatively high activity of plantarflexor on the non-paretic side and in healthy controls.