CONTEXT Greater hamstrings stiffness is associated with less anterior tibial translation during controlled perturbations. However, it is unclear how hamstrings stiffness influences anterior cruciate ligament (ACL) loading mechanisms during dynamic tasks. OBJECTIVE To evaluate the influence of hamstrings stiffness on landing biomechanics related to ACL injury. DESIGN Cross-sectional study. SETTING Research laboratory. PATIENTS OR OTHER PARTICIPANTS A total of 36 healthy, physically active volunteers (18 men, 18 women; age = 23 ± 3 years, height = 1.8 ± 0.1 m, mass = 73.1 ± 16.6 kg). INTERVENTION(S) Hamstrings stiffness was quantified via the damped oscillatory technique. Three-dimensional lower extremity kinematics and kinetics were captured during a double-legged jump-landing task via a 3-dimensional motion-capture system interfaced with a force plate. Landing biomechanics were compared between groups displaying high and low hamstrings stiffness via independent-samples t tests. MAIN OUTCOME MEASURE(S) Hamstrings stiffness was normalized to body mass (N/m·kg(-1)). Peak knee-flexion and -valgus angles, vertical and posterior ground reaction forces, anterior tibial shear force, internal knee-extension and -varus moments, and knee-flexion angles at the instants of each peak kinetic variable were identified during the landing task. Forces were normalized to body weight, whereas moments were normalized to the product of weight and height. RESULTS Internal knee-varus moment was 3.6 times smaller in the high-stiffness group (t22 = 2.221, P = .02). A trend in the data also indicated that peak anterior tibial shear force was 1.1 times smaller in the high-stiffness group (t22 = 1.537, P = .07). The high-stiffness group also demonstrated greater knee flexion at the instants of peak anterior tibial shear force and internal knee-extension and -varus moments (t22 range = 1.729-2.224, P < .05). CONCLUSIONS Greater hamstrings stiffness was associated with landing biomechanics consistent with less ACL loading and injury risk. Musculotendinous stiffness is a modifiable characteristic; thus exercises that enhance hamstrings stiffness may be important additions to ACL injury-prevention programs.