Electrical stimulation of the spinal cord (ESSC) is a method enabling researchers to confirm the existence of the stepping movement generator in humans. Currently, the ESSC-based technologies of motor rehabilitation of patients immobilized after spinal cord injury are actively developing. The impact of the serotonergic system in the organization of movement is intensively studied both at the systemic and the molecular–cellular level in a large number of researches. The aim of this review was to analyze the current experimental data on the participation of serotonergic system in the locomotor activity control at the spinal level with a focus on the processes related to electrical stimulation of spinal locomotor networks. Special interest is given to the serotonin-based regulation of human motor activity. The data on the use of serotonin-modulating pharmacotherapy for increasing the efficacy of the ESSC method in locomotor rehabilitation are presented in the final part of the review.