While the spring-like leg behavior of legs in mammalian locomotion has been well documented, its neural basis remains ambiguous. The purpose of the present study was to examine leg stiffness control during hopping. Seven male subjects performed in place two-legged hopping at their preferred frequency with two different contact times of the stance phase, preferred and short ones (PCT and SCT, respectively). Based on a spring-mass model, leg stiffness was calculated from the subjects' body mass, ground contact and flight times. Surface electromyographic (EMG) activities of the medial gastrocnemius (MG), soleus (SOL) and tibialis anterior (TA) muscles were recorded. Leg stiffness was higher in the SCT condition than in the PCT condition. The SCT condition was characterized by high EMG activity of MG and SOL at both pre- and post-landing phases, which peaked at about 50 ms. On the other hand, the activity of TA was low throughout the contact phase as compared with those of MG and SOL, and its peak value around 50 ms after landing was significantly lower for the SCT condition than for the PCT condition. We conclude that (1) the leg stiffness is regulated by a change in centrally programmed muscle preactivation and probably also by a concomitant change in the short-latency stretch reflex response of the triceps surae muscles, and (2) the co-contraction of antagonistic TA does not play a major role in leg stiffness control.