The aim of this study was to examine the effect of vibration on motor unit (MU) firing behavior and physical performance of antagonist muscles in healthy young adult males. Fourteen males (age = 24.3 ± 3.6years) were included in this study. There were two conditions, one in which participants received 80Hz vibration in the distal tendon of the hamstring for 30s and the control condition (no vibration). High-density surface electromyography (HD-SEMG) signals and maximal voluntary contraction (MVC) of knee extensor muscles were evaluated before and after the respective conditions and recorded from the vastus lateralis muscle during submaximal ramp-up and sustained contractions at 30% MVC. Convolution blind source separation was used to decompose the HD-SEMG signals into individual MU firing behaviors. In total, 739 MUs were detected (control; 360 MUs and vibration; 379 MUs), and a total of 312 matched MUs were identified across both submaximal contraction conditions (control: 150 MUs; vibration: 162 MUs). Vibration significantly increased the discharge rate (p = 0.047) and decreased the recruitment threshold before and after intervention (p = 0.001) but not in the control condition. Furthermore, the recruitment threshold is a factor that influences discharge rate. Significant correlations were observed between the recruitment threshold and both the ∆ discharge rate and the ∆ recruitment threshold under the vibration condition (p < 0.001). Vibration increased in the discharge rate and decreased the recruitment threshold of the antagonist muscle. These findings suggested that vibration contributes to immediate changes in the neural control of antagonist muscles.