The aim of this study was to determine if botulinum toxin type A (BoNT-A) injection into the medial hamstring can improve gait kinematics and muscle-tendon length in spastic cerebral palsy (CP) with a flexed knee gait (FKG). Twenty-nine children with spastic CP (Gross Motor Function Classification System I–III) with FKG were recruited for this prospective study. BoNT-A was injected into the semitendinosus and semimembranosus (SM) muscles under ultrasonography guidance. Assessments included Gross Motor Function Measure (GMFM), Modified Ashworth Scale (MAS), Modified Tardieu Scale (MTS), 3-dimensional computerized gait analysis, calculated SM muscle-tendon length and lengthening velocity during gait using musculoskeletal modeling at baseline, 4 and 16 weeks after the injection. Compared to baseline data, significant improvements in GMFM, MAS, and MTS were demonstrated at weeks 4 and 16, and also a significant increase in maximum knee extension during the stance phase was observed at week 4. In addition, the mean lengthening velocity during the swing phase was increased at week 16 without a change in the SM muscle length. Furthermore, there was a significant increase in anterior pelvic tilt at week 4, compared to baseline data. The significant decrease in hip internal rotation after injection was observed only in children with excessive hip internal rotation at initial contact before injection. BoNT-A injection into hamstrings leads to a significant increase in knee extension and anterior pelvic tilt with an increase in lengthening velocity of SM in spastic CP with FKG.