Neuronal networks within the spinal cord, collectively known as the central pattern generator (CPG), coordinate rhythmic movements underlying locomotion. The transcription factor doublesex and mab-3-related transcription factor 3 (DMRT3) is involved in the differentiation of the dorsal interneuron 6 class of spinal cord interneurons. In horses, a non-sense mutation in theDmrt3gene has major effects on gaiting ability, whereas mice lacking theDmrt3gene display impaired locomotor activity. Although theDmrt3gene is necessary for normal spinal network formation and function in mice, a direct role forDmrt3-derived neurons in locomotor-related activities has not been demonstrated. Here we present the characteristics of theDmrt3-derived spinal cord interneurons. Using transgenic mice of both sexes, we characterized interneurons labeled by their expression of Cre driven by the endogenousDmrt3promoter. We used molecular, retrograde tracing and electrophysiological techniques to examine the anatomical, morphological, and electrical properties of the Dmrt3-Cre neurons. We demonstrate that inhibitory Dmrt3-Cre neurons receive extensive synaptic inputs, innervate surrounding CPG neurons, intrinsically regulate CPG neuron's electrical activity, and are rhythmically active during fictive locomotion, bursting at frequencies independent to the ventral root output. The present study provides novel insights on the character of spinalDmrt3-derived neurons, data demonstrating that these neurons participate in locomotor coordination.SIGNIFICANCE STATEMENTIn this work, we provide evidence for a role of the Dmrt3 interneurons in spinal cord locomotor circuits as well as molecular and functional insights on the cellular and microcircuit level of the Dmrt3-expressing neurons in the spinal cord. Dmrt3 neurons provide the first example of an interneuron population displaying different oscillation frequencies. This study presents novel findings on an under-reported population of spinal cord neurons, which will aid in deciphering the locomotor network and will facilitate the design and development of therapeutics for spinal cord injury and motor disorders.