Altered semitendinosus (ST) morphology and distal tendon insertion following anterior cruciate ligament reconstruction (ACLR) may reduce knee flexion torque generating capacity of the hamstrings via impaired ST force generation and/or moment arm. This study used a computational musculoskeletal model to simulate mechanical consequences of tendon harvest for ACLR on ST function by modeling changes in ST muscle tendon insertion point, moment arm, and torque generating capacity across a physiological range of motion. Simulated ST function was then compared between ACLR and uninjured contralateral limbs. Magnetic resonance imaging from 18 individuals with unilateral history of ACLR involving a hamstring autograft was used to analyse bilateral hamstring muscle (ST, semimembranosus, bicep femoris long head and short head) morphology and distal ST tendon insertion. The ACLR cohort was sub-grouped into those with and without ST regeneration. For each participant with ST regeneration (n = 7), a personalized musculoskeletal model was created including postoperative remodeling of ST using OpenSim 4.1. Knee flexion and internal rotation moment arms and torque generating capacities of hamstrings were evaluated. Bilateral differences were calculated with an asymmetry index (%) ([unaffected limb–affected limb]/[unaffected limb + affected limb]*100%). Smaller moment arms or knee torques within injured compared to uninjured contralateral limbs were considered a deficit. Compared to uninjured contralateral limbs, ACLR limbs with tendon regeneration (n = 7) had minor reductions in knee flexion (5.80% [95% confidence interval (CI) = 3.97–7.62]) and internal rotation (4.92% [95% CI = 2.77–7.07]) moment arms. Decoupled from muscle morphology, altered ST moment arms in ACLR limbs with tendon regeneration resulted in negligible deficits in knee flexion (1.20% [95% CI = 0.34–2.06]) and internal rotation (0.24% [95% CI = 0.22–0.26]) torque generating capacity compared to uninjured contralateral limbs. Coupled with muscle morphology, ACLR limbs with tendon regeneration had substantial deficits in knee flexion (19.32% [95% CI = 18.35–20.28]) and internal rotation (15.49% [95% CI = 14.56–16.41]) torques compared to uninjured contralateral limbs. Personalized musculoskeletal models with measures of ST distal insertion and muscle morphology provided unique insights into post-ACLR ST and hamstring function. Deficits in knee flexor and internal rotation moment arms and torque generating capacities were evident in those with ACLR even when tendon regeneration occurred. Future studies may wish to implement this framework in personalized musculoskeletal models following ACLR to better understand individual muscle function for injury prevention and treatment evaluation.