Inhibitory neurons are an essential element of the locomotor network in the mammalian spinal cord. However, little is known about the firing pattern and synaptic modulation during locomotion in the majority of them. In this study, we performed whole cell recording in visually identified ventrolaterally located GABAergic neurons (VL-GNs) in the rostral (L2 segment) and caudal (L5 segment) lumbar cord using isolated spinal cord preparations taken from glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse neonates. These neurons did not respond to electrical stimulation of the ventral root, indicating that they were not Renshaw cells. Ninety-five percent of VL-GNs in the L2 segment and fifty percent of those in the L5 segment showed significant rhythmic firing during locomotor-like rhythmic activity induced by bath application of 5-HT and NMDA. Seventy percent of these neurons fired mainly during the extensor phase, and twenty-five percent fired mainly during the flexor phase. Voltage-clamp recordings revealed that most of these neurons received rhythmic inhibition during the nonfiring phase and excitatory synaptic inputs during the firing phase. Morphological examination of recorded neurons filled with neurobiotin showed that their soma was located lateral to the motoneuron pool and that they extended their processes into the local ipsilateral ventromedial region and dorsal regions. The present study indicates that these GABAergic interneurons located in the ventrolateral region adjacent to the motoneuron pool are rhythmically active during locomotion and involved in the inhibitory modulation of local locomotor network in the lumbar spinal cord.