After spinal cord transection, the generation of stepping depends on neurotransmitter systems entirely contained within the local lumbar spinal cord. Glutamate and glycine likely play important roles, but surprisingly little is known about how the content of these two key neurotransmitters changes to achieve weight-bearing stepping after spinal cord injury. We studied the levels of glutamate and glycine in the lumbar spinal cord of spinally transected rats. Rats (n = 48) received spinal cord transection at 5 days of age, and 4 weeks later half were trained to step using a robotic treadmill system and the remaining half were untrained controls. Analyses of glutamate and glycine content via high-performance liquid chromatography showed training significantly raised the levels of both neurotransmitters in the lumbar spinal cord beyond normal. The levels of both neurotransmitters were significantly correlated with the ability to perform independent stepping during training. Glutamate and glycine levels were not significantly different between Untrained and Normal rats or between Trained and Untrained rats. There was a trend for higher expression of VGLUT1 and GLYT2 around motor neurons in Trained versus Untrained rats based on immunohistochemical analyses. Training improved the ability to generate stepping at a range of weight support levels, but normal stepping characteristics were not restored. These findings suggested that the remodeling of the lumbar spinal circuitry in Trained spinally transected rats involved adaptations in the glutamatergic and glycinergic neurotransmitter systems. These adaptations may contribute to the generation of novel gait patterns following complete spinal cord transection.