Sensory afferents to the spinal cord is the main source for therapeutic intervention and rehabilitation that can generate and coordinate complex movements even without supraspinal control. Using the activity-dependent marker c-fos, we identified cholinergic lumbar interneurons around the central canal that are activated during load bearing stepping in intact and complete spinal cord transected adult rats. We observed activation of cholinergic central canal cluster and partition neurons in intact rats after quadrapedal stepping and in spinal rats step trained bipedally for 5 weeks with epidural and pharmacological stimulation. Furthermore, increased load imposed during treadmill stepping activated more cholinergic interneurons in adult rats. Intact adult mice whose tibialis anterior (TA) hindlimb muscle was injected with transynaptic pseudorabies virus, showed activated cholinergic interneurons in the TA spinal circuitry after quadrapedal stepping. These data demonstrate the role of sensorimotor pathways and specific interneurons in the recovery after spinal cord injury.