Background: Ambulation is an essential aspect of daily living and is often impaired after brain and spinal cord injuries. Despite the implementation of standard neurorehabilitative care, locomotor recovery is often incomplete. Objective: In this randomized, sham-controlled, double-blind, parallel design study, we aimed to determine if anodal transcutaneous spinal direct current stimulation (anodal tsDCS) could improve training effects on locomotion compared to sham (sham tsDCS) in healthy subjects. Methods: 43 participants underwent a single backwards locomotion training (BLT) session on a reverse treadmill with concurrent anodal (n=22) or sham (n=21) tsDCS. The primary outcome measure was speed gain measured 24 hours post-training. We hypothesized that anodal tsDCS+BLT would improve training effects on backward locomotor speed compared to sham tsDCS+BLT. A subset of participants (n=31) returned for two additional training days of either anodal (n=16) or sham (n=15) tsDCS and underwent (n=29) H-reflex testing immediately before, immediately after, and 30 minutes post-training over three consecutive days. Results: A single session of anodal tsDCS+BLT elicited greater speed gain at 24 hours relative to sham tsDCS+BLT (p=0.008, two-sample t-test, adjusted for one interim analysis after the initial 12 subjects). Anodal tsDCS+BLT resulted in higher retention of the acquired skill at day 30 relative to sham tsDCS+BLT (p=0.002) in the absence of significant group differences in online or offline learning over the three training days (p=0.467 and p=0.131). BLT resulted in transient down-regulation of H-reflex amplitude (Hmax/Mmax) in both test groups (p<0.0001). However, the concurrent application of anodal-tsDCS with BLT elicited a longer lasting effect than sham-tsDCS+BLT (p=0.050). Conclusion: TsDCS improved locomotor skill acquisition and retention in healthy subjects and prolonged the physiological exercise-mediated downregulation of excitability of the alpha motoneuron pool. These results suggest that this strategy is worth exploring in neurorehabilitation of locomotor function.