Cerebral palsy (CP) is the most common motor disability in childhood. Children with CP often develop abnormal gait patterns such as a shorter and slower step, a limited range-of-motion of the ankle, knee, and hip joints, and abnormal muscle spasticity and activation patterns. Whole-body vibration (WBV) is a new intervention paradigm that has demonstrated its effectiveness in clinical populations such as adults with stroke and children with CP. However, the effect of WBV frequency and amplitude and the optimal intervention dosage have not been fully understood in children with CP. The purpose of this study was to evaluate the acute effect of single-session WBV with two different amplitudes on (a) muscle spasticity, spatiotemporal gait parameters, and standing posture, and (b) joint kinematics and muscle activation patterns during walking in children with CP. Ten children with spastic CP aged 7-17 years participated in this study. Two WBV sessions were presented with the same frequency of 20Hz but two amplitudes (low amplitude: 1mm and high amplitude: 2mm). Each vibration session included 6 sets of 90-second vibration exposure and 90-second rest. Modified Ashworth scale, overground walking, and 60-second quiet standing tasks were performed at baseline and after each WBV session. Four muscles were studied including lateral gastrocnemius, tibialis anterior, vastus lateralis, and biceps femoris from the affected side for those with hemiplegia diagnosis and from the less affected side for those with diplegia diagnosis. For data analysis, participants were categorized into either a high-response (increasing stride length after WBV intervention) or a low-response subgroup (no change in stride length). Both subgroups reduced spasticity after both WBV sessions, and the high-response subgroup displayed a decreased postural sway during standing after the high-amplitude WBV. Both subgroups, especially the high-response subgroup, displayed increased ankle range-of-motion and decreased muscle activity during overground walking, which might be due to the reduced spasticity. Our results suggest that a single-session of WBV intervention with a higher amplitude can induce an acute effect on reducing muscle spasticity and improving motor function in some children with CP.