After hindlimb unloading (HU), the adaptive changing of the rat step cycle duration, kinematics of the ankle and knee joints, and duration of one-joint ankle extensor m. soleus (SOL) activity are detected. However, how the activity of their synergist gastrocnemius medialis muscle (GM) changes in locomotion after HU remains unknown. GM is a two-joint muscle that produces both extension and flexion torques at the ankle and knee, respectively, regardless of the step cycle phase. The aim of our study was to assess changes in the flexor and extensor activity of GM and their influence on hindlimb kinematics after HU. The hindlimb kinematics, activity of GM, and SOL were evaluated, and semitendinosus muscle (ST) activity was registered in six Wistar rats in treadmill locomotion before and after HU. The mean EMG of the GM activity, which was co-active with ST burst activity, significantly increased after HU. The mean EMG of the GM activity, which was co-active with SOL activity, was unchanged after HU, but both SOL and GM bursts had a tendency to increase in duration. Hyperextension of the knee joint and the tendency to overextension of the ankle joint in the late of the stance phase were revealed after HU. The results show that the absence of weight bearing leads to an increase only in the flexor activity of GM and does not affect the extensor GM activity. Possible mechanisms of changes in GM activity and joint kinematics after HU are discussed.