Objective To study the effect of gender and maneuvers on anterior cruciate ligament (ACL) injury risk factors for volleyball players. Methods Sports biomechanics data of volleyball players during stop-jump, drop landing and sidestep cutting were collected. The ACL injury rate and biomechanical parameters of simulated injured jumps were obtained with Monte Carlo simulation. The influence of gender and maneuvers on ACL injury risk factors was validated by 2×3 mixed designed two-way ANOVA. Results Sidestep cutting was the highest risk maneuver of ACL injury for both genders (P＜0.001). Compared with male players, female players had a greater risk of ACL injury during sidestep cutting and stop-jump (P＜0.001), while male players were more prone to have ACL injury than female players during drop landing (P＜0.001). The risk factors of ACL injury obtained by simulation were significantly influenced by gender and maneuvers (P＜0.001). Conclusions Male players were more likely to increase ACL load due to smaller knee flexion, forward leg tilt and heel landing than female players during sidestep cutting, while female players owned larger ground reaction force (GRF) and knee extension moment. Smaller knee flexion angle during stop jump was the major risk factor for both genders, however more characteristics contributed to the males. Female players with large GRF, knee valgus and extension moment, and heel-landing were likely to have ACL injury, while the small knee flexion angle was the key risk factor for male players. The results can provide evidences for evaluation of volleyball players’ ACL injury risk, individualized injury prevention protocols, and clinical treatment and rehabilitation directions.