The spinal cord contains neural circuits that can produce the rhythm and pattern of locomotor activity. It has previously been postulated that a rhythmogenic population of glutamatergic neurons, termed Hb9 interneurons, contributes to this rhythmogenesis. The homeobox gene, Hb9, is expressed in these interneurons as well as motor neurons. We developed a mouse line in which cre recombinase activity is inducible in neurons expressing Hb9. We then used this line to eliminate vesicular glutamate transporter 2 from Hb9 interneurons, and found that there were no deficits in treadmill locomotion. We conclude that glutamatergic neurotransmission by Hb9 interneurons is not required for locomotor rhythmogenesis. The role of these neurons in neural circuits remains elusive.