The aim of this study was to identify the contribution of the Soleus and Gastrocnemius (Gastroc) muscles' forces to anterior cruciate ligament (ACL) loading during single-leg landing. Although Quadriceps (Quads) and Hamstrings (Hams) muscles were recognized as the main contributors to the ACL loading, less is known regarding the role of ankle joint plantarflexors during landing. Eight healthy subjects performed single-landing tasks from 30 and 60cm heights. Scaled generic musculoskeletal models were developed in OpenSim to calculate lower limb muscle forces. The model consisted of 10 segments with 23 degrees of freedom and 92 lower body muscle-tendon units. Knee joint reaction forces were calculated based on the estimated muscle forces and used to predict ACL forces. We hypothesized that Soleus and Gastrocs muscle forces have opposite effects on tibial loading in the anterior/posterior directions. In situations where greater landing height would lead to an increase in GRF and risk of ACL injury, we further hypothesized that posterior forces of the Soleus and Hams would increase correspondingly to help protect the ACL during a safe landing maneuver. Our results demonstrated the antagonistic and agonistic roles of Gastrocs and Soleus respectively in ACL loading. The posterior force of Soleus reached 28-32% of Ham's posterior force for both landing heights at peak GRF while the posterior force of Gastrocs on femur was negligible. ACL injury risk during single-leg landing is not only dependent on knee musculature but also influenced by muscles that do not span the knee joint, such as the Soleus. In conclusion, the role of the ankle plantarflexors should be considered when developing training strategies for ACL injury prevention.