This study examined ten human adult cadaveric knees to dissect the popliteus muscle-tendon unit (PMTU), including its numerous attachments to other posterior and posterolateral structures, and to determine the effect of tensioning the PMTU on the internal and external rotation, total rotational arcs, and neutral tibial rotation in full extension and 30 degrees, 60 degrees, and 90 degrees of knee flexion. The junction between the popliteus tendon and the fibular head commonly described as the popliteofibular ligament became lax in internal and tense in external tibial rotation. The internal and external rotational arcs increased gradually between extension and 90 degrees of flexion. Tensioning the PMTU resulted in a statistically highly significant internal tibial rotation with decreased internal and increased rotational arcs. The anatomical findings and functional data indicate that the PMTU is an important structure maintaining dorsolateral stability, stabilizing the lateral meniscus, and balancing the neutral tibial rotation.