An erect posture with bipedal locomotion is a characteristic feature of humans compared to other mammals. Most of our knowledge about the hierarchical network of supraspinal locomotor centers derives from animal experiments, mainly in the cat. We posed the question of whether evolutionary transition from quadrupedal to bipedal locomotion – with associated change of foreleg function – caused reorganization of these supraspinal locomotor centers. Using functional magnetic resonance imaging, we identified separate and distinct cerebellar and brainstem BOLD signal increases related to posture and gait during mental imagery of standing, walking, and running in healthy volunteers ( n = 26). Comparison with the locomotion centers in the cat showed that these activations include the pacemakers for gait initiation and speed regulation in the interfastigial cerebellum and bilateral midbrain tegmentum (cerebellar and mesencephalic locomotor regions), their descending target regions in the pontine reticular formation, and the rhythm generators in the cerebellar vermis and paravermal cortex. Moreover, during mental imagery of stance, a BOLD signal increase was observed in the dorsal pons, reflecting an activation of the dorsal tegmental field, a locomotion-suppressing site in the cat. These results support the view that the organization of supraspinal locomotor centers was preserved during the transition to bipedal locomotion. The clinical relevance of these centers has so far been largely neglected. However, Parkinson's disease, for example, is associated with reduced cell counts in the pedunculopontine nucleus, a part of the mesencephalic locomotor region. This association suggests that deep brain stimulation of locomotion centers may provide new therapeutic approaches for common gait disorders.