© 2022, The Author(s), under exclusive licence to Royal Academy of Medicine in Ireland.Background: In spite of treadmill training and multilevel botulinum toxin (BoNT-A) injection being the two most commonly used treatment methods in pediatric rehabilitation management, there was no study investigating the effect of treadmill training after BoNT-A injection in children with cerebral palsy (CP). Aim: The aim of this study was to investigate the effect of treadmill training in addition to routine physical therapy after BoNT-A injection in ambulatory children with spastic bilateral CP on lower extremity muscle strength, selective motor control, and mobility. Methods: A total of 30 spastic bilateral children with CP classified level II–III by the Gross Motor Function Classification System were randomly assigned the study and control groups. Both groups continued routine physical therapy treatments after multilevel BoNT-A injection into lower extremities, while the study group additionally underwent 8 weeks of treadmill training (20 min, two sessions per week). Handheld dynamometer, selective control assessment of lower extremity, temporospatial evaluation of gait, and Pediatric Evaluation of Disability Inventory were assessed before and after 8 weeks. Results: In both groups, hip, knee, and ankle muscle strength increased at the end of 8 weeks (p < 0.05); however, in the study group, hip flexor/extensor muscle strength (p < 0.05, ES ≥ 0.50), selective motor control of ankle (p < 0.01, ES = 1.17), walking speed (p < 0.01, ES = 2.60), step lengths (p < 0.01, ES = 1.32), and mobility (p < 0.01, ES = 1.37) increased significantly compared to those of the control group. Conclusions: Treadmill training in addition to routine physical therapy after BoNT-A injection is beneficial for hip muscle strength, ankle selective motor control, walking quality, and functional mobility in the short term. Trial registration: ClinicalTrials.gov identifier: NCT03580174.