Background: Anterior cruciate ligament (ACL) injury reduction training has focused on lower body strengthening and landing stabilization. In vitro studies have shown that quadriceps forces increase ACL strain, and hamstring forces decrease ACL strain. However, the magnitude of the effect of the quadriceps and hamstrings forces on ACL loading and its timing during in vivo landings remains unclear. Purpose: To investigate the effect and timing of knee muscle forces on ACL loading during landing. Study Design: Descriptive laboratory study. Methods: A total of 13 young female athletes performed drop vertical jump trials, and their movements were recorded with 3-dimensional motion capture. Lower limb joint motion and muscle forces were estimated with OpenSim and applied to a musculoskeletal finite element (FE) model to estimate ACL loading during landings. The FE simulations were performed with 5 different conditions that included/excluded kinematics, ground-reaction force (GRF), and muscle forces. Results: Simulation of landing kinematics without GRF or muscle forces yielded an estimated median ACL strain and force of 5.1% and 282.6 N. Addition of GRF to kinematic simulations increased ACL strain and force to 6.8% and 418.4 N ( P < .05). Addition of quadriceps force to kinematics + GRF simulations nonsignificantly increased ACL strain and force to 7.2% and 478.5 N. Addition of hamstrings force to kinematics + GRF simulations decreased ACL strain and force to 2.6% and 171.4 N ( P < .001). Addition of all muscles to kinematics + GRF simulations decreased ACL strain and force to 3.3% and 195.1 N ( P < .001). With hamstrings force, ACL loading decreased from initial contact (time of peak: 1-18 milliseconds) while ACL loading without hamstrings force peaked at 47 to 98 milliseconds after initial contact ( P = .024-.001). The knee flexion angle increased from 20.9° to 73.1° within 100 milliseconds after initial contact. Conclusion: Hamstrings activation had greater effect relative to GRF and quadriceps activation on ACL loading, which significantly decreased and regulated the magnitude and timing of ACL loading during in vivo landings. Clinical Relevance: Clinical training should focus on strategies that influence increased hamstrings activation during landing to reduce ACL loads.