Locomotion requires the continuous integration of descending motor commands and sensory inputs from the legs by spinal central pattern generator circuits. Modulation of spinal neural circuits by transspinal stimulation is well documented, but how transspinal stimulation affects corticospinal excitability during walking in humans remains elusive. We measured the motor evoked potentials (MEPs) at multiple phases of the step cycle conditioned with transspinal stimulation delivered at sub- and suprathreshold intensities of the spinally mediated transspinal evoked potential (TEP). Transspinal stimulation was delivered before or after transcranial magnetic stimulation during which summation between MEP and TEP responses in the surface EMG was absent or present. Relationships between MEP amplitude and background EMG activity, silent period duration, and phase-dependent EMG amplitude modulation during and after stimulation were also determined. Ankle flexor and extensor MEPs were depressed by suprathreshold transspinal stimulation when descending volleys were timed to interact with transspinal stimulation-induced motoneuron depolarization at the spinal cord. MEP depression coincided with decreased MEP gain, unaltered MEP threshold, and unaltered silent period duration. Locomotor EMG activity of bilateral knee and ankle muscles was significantly depressed during the step at which transspinal stimulation was delivered but fully recovered at the subsequent step. The results support a model in which MEP depression by transspinal stimulation occurs via subcortical or spinal mechanisms. Transspinal stimulation disrupts the locomotor output of flexor and extensor motoneurons initially, but the intact nervous system has the ability to rapidly overcome this pronounced locomotor adaptation. In conclusion, transspinal stimulation directly affects spinal locomotor centers in healthy humans. Lumbar transspinal stimulation decreases ankle flexor and extensor motor evoked potentials (MEPs) during walking. The MEP depression coincides with decreased MEP gain, unaltered MEP threshold changes, and unaltered silent period duration. These findings indicate that MEP depression is subcortical or spinal in origin. Healthy subjects could rapidly overcome the pronounced depression of muscle activity during the step at which transspinal stimulation was delivered. Thus, transspinal stimulation directly affects the function of spinal locomotor networks in healthy humans.