Understanding the distinct functions of human muscles could not only help professionals obtain insights into the underlying mechanisms that we accommodate compromised neuromuscular system, but also assist engineers in developing rehabilitation devices. This study aims to determine the contribution of major muscle and the energy flow in the human musculoskeletal system at four sub-phases (collision, rebound, preload, push-off) during the stance of walking at different speeds. Gait experiments were performed with three self-selected speeds: slow, normal, and fast. Muscle forces and mechanical work were calculated by using a subject-specified musculoskeletal model. The functions of individual muscles were characterized as four functional behaviors (strut, spring, motor, damper), which were determined based on the mechanical energy. The results showed that during collision, hip flexors (iliacus and psoas major) and ankle dorsiflexors (anterior tibialis) were the most dominant muscles in buffering the stride with energy absorption; during rebound, the posterior muscles (gluteus maximus, gastrocnemius, posterior tibialis, soleus) contributed the most to energy generation; during preload, energy for preparing push-off was mainly absorbed by the muscles surrounding knee (vastus, semimembranosus, semitendinosus); during push-off, ankle plantar flexors (gastrocnemius, soleus, posterior tibialis, peroneus muscles, flexor digitorum, flexor hallucis) mainly behaved to generate energy for forward propulsion. With increased walking speed, additional energy (almost 400%) from harder stride was mainly absorbed by the flexor muscles. Hip extensors and adductors transferred more energy (around 150%) to the distal segments during rebound. Soleus and gastrocnemius muscles generated more energy (about 75%) to the proximal segments for propulsion. Along with our previous study of joint-level energy analysis, these findings could assist better understanding of human musculoskeletal behaviors during locomotion and provide principles for the bio-design of related assistive devices from motors performance enhancement to rehabilitation such as exoskeleton and prosthesis.