Objective The purposes of study were to verify the effect of gender and maneuvers on Anterior Cruciate Ligament (ACL) injuries risk factors for volleyball players with Monte Carlo simulation. Methods Kinematics, kinetic and EMG were collected for volleyball players during stop-jump, drop landing and sidestep cutting maneuvers. The ACL injury rate and lower extremity biomechanical characteristics of simulated injured jumps were output with Simulation. 2×3 mixed designed two-way ANOVA was used to identify the influence of gender and maneuvers on ACL injuries risk factors. Results Sidestep cutting was the highest risk maneuver of ACL injury for both genders (P<0.001). Compared to males, females 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 females 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). Conclusion Male players were more likely to increase ACL load due to smaller knee flexion, forward leg tilt and heel landing than females during sidestep cutting, while females owned larger GRF and knee extension moment. The risk factors for female players was smaller knee flexion angle during stop jump, however the forward leg tilt, heel landing, larger GRF, knee valgus and extension moment contributed to male athletes ACL injuries. Female players with large posterior and vertical 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 males. The results could provide evidences for evaluation of volleyball players’ ACL injury risk, individualized injury prevention protocols, and clinical treatment and rehabilitation directions.