Genu valgum (GV) is a common lower limb deformity that may increase the risk of anterior cruciate ligament (ACL) injury. This study used OpenSim musculoskeletal modeling and kinematic analysis to investigate the mechanical responses of the ACL under fatigue in females with GV. Eight females with GV and eight healthy controls completed a running-induced fatigue protocol. Lower limb kinematic and kinetic data were collected and used to simulate stress and strain in the anteromedial ACL (A–ACL) and posterolateral ACL (P–ACL) bundles, as well as peak joint angles and knee joint stiffness. The results showed a significant interaction effect between group and fatigue condition on A–ACL stress. In the GV group, A–ACL stress was significantly higher than in the healthy group both before and after fatigue (p < 0.001) and further increased following fatigue (p < 0.001). In the pre-fatigued state, A–ACL strain was significantly higher during the late stance phase in the GV group (p = 0.036), while P–ACL strain significantly decreased post-fatigue (p = 0.005). Additionally, post-fatigue peak hip extension and knee flexion angles, as well as pre-fatigue knee abduction angles, showed significant differences between groups. Fatigue also led to substantial changes in knee flexion, adduction, abduction, and hip/knee external rotation angles within the GV group. Notably, knee joint stiffness in this group was significantly lower than in controls and decreased further post-fatigue. These findings suggest that the structural characteristics of GV, combined with exercise-induced fatigue, exacerbate A–ACL loading and compromise knee joint stability, indicating a higher risk of ACL injury in fatigued females with GV.