Traumatic spinal cord injury is a devastating neurologic condition in both veterinary and human medicine and despite research yielding numerous potential interventions with remarkable efficacy demonstrated in rodent models, none has advanced to successful clinical translation. Pet dogs’ predilection for sustaining spinal cord injury, typically due to intervertebral disc herniation or vertebral column fracture, makes them a suitable clinical model in which putative interventions for spinal cord injury can be tested. In recent years, there has been a growing body of experimental evidence that attests to the efficacy of chondroitinase ABC in promoting axonal regeneration and functional recovery after spinal cord injury by reactivating neuroplasticity. Chondroitinase ABC is a commercialized bacterial enzyme that selectively deglycosylates chondroitin sulfate proteoglycans and thereby disrupts the perineuronal nets that limits axonal regeneration. In this 60-dog clinical trial, we examined the therapeutic effect of chondroitinase ABC on the primary outcome measure, pelvic-thoracic limb gait coordination, and several secondary parameters, including motor- and sensory-evoked potentials and urinary bladder compliance. While our study failed to detect a therapeutic effect in chronic, severe thoracolumbar spinal-injured dogs, it has established drug safety in a clinical large animal model. Thus, this study has provided a platform for future investigations in which the dose, route and timing of chondroitinase ABC administration, as well as patient selection, can be adjusted to maximize its potential therapeutic effect and benefit spinal-injured human and veterinary patients, especially if a more treatment-responsive subgroup could be identified.