Introduction: The spike is not only an active technique to score, but increases the probability of winning. Although, the spike is considered a better offensive skill, that results in a higher injury rate than other technique in volleyball competition. Volleyball competition is a non-contact sport, but it had a high musculoskeletal injury rate after landing movement. A lot of volleyball players had lower extremity injuries which often occurred after landing movement, and the females possessed a greater non-contact ACL injury rate than males in the same competitions. Landing movement is one of the primary factor for injury in female. The aim of this study was to investigate the biomechanical differences between male and female players after a volleyball spike landing. Methods: Eight male and 8 female volleyball players performed spike landing on two force plates (1500Hz) with Vicon motion analysis system (10 cameras, 300Hz) to collect the kinetic and kinematic data. An independent t-test was used to test the kinematic and kinetic variables differences between male and female volleyball players. Results: There was no significant difference in ground reaction force between male and female players. In the sagittal plane motion, males exhibited greater hip flexion angles at the initial ground contact. Peak hip flexion angles and hip range of motion (ROM) were greater in males. Females exhibited greater peak ankle dorsiflexion angles and ankle ROM. Males exhibited greater hip angles in the peak vertical ground reaction force (PVGRF). In the frontal plane motion, females exhibited greater knee valgus angles in the initial ground contact and PVGRF during landing. Peak knee valgus angles were greater in females. Conclusions: Genders displayed different movement during volleyball spike landing. Males sufficiently utilized hip joint motion to perform spike landing that might reduce more ground impact. Females mainly utilized ankle motion to perform spike landing, and accompanied with a greater knee valgus angles than males during the spike landing, which might increase risk of injury.