Parkinson’s disease is associated with hyperactivity of the subthalamic nucleus (STN), contributing to motor and gait disturbances. Although deep brain stimulation of the STN alleviates certain motor dysfunction, its specific effect on gait abnormalities remains controversial. This study investigated the long-term changes in locomotion following direct infusions of botulinum toxin-A into the globus pallidus internal segment (GPi) to suppress the flow of information from the STN to the GPi in a hemiparkinsonian rat model. Static and dynamic gait parameters were quantified using a CatWalk apparatus. Interestingly, botulinum toxin-A at 0.5 ng significantly reduced only the dynamic gait parameters of hemiparkinsonian rats at 1 week and 1 month post-infusion, while static gait parameters did not change. This study offers new insights into the complexity of basal ganglia in locomotor control and shows the potential of central infusion of botulinum toxin-A as a novel intervention in the study of experimental hemiparkinson’s disease.