Motor behaviors result from the interplay between the brain and the spinal cord. Reticulospinal neurons, situated between the supraspinal structures that initiate motor movements and the spinal cord that executes them, play key integrative roles in these behaviors. However, the molecular identities of mammalian reticular formation neurons that mediate motor behaviors have not yet been determined, thus limiting their study in health and disease. In the medullary reticular formation of the mouse, we identified neurons that express the transcription factors Lhx3 and/or Chx10, and demonstrate that these neurons form a significant component of glutamatergic reticulospinal pathways. Lhx3-positive medullary reticular formation neurons express Fos following a locomotor task in the adult, indicating that they are active during walking. Furthermore, they receive functional inputs from the mesencephalic locomotor region and have electrophysiological properties to support tonic repetitive firing, both of which are necessary for neurons that mediate the descending command for locomotion. Together, these results suggest that Lhx3/Chx10 medullary reticular formation neurons are involved in locomotion.