The effect of the topical application of human amniotic fluid (HAF) on peritendinous adhesion formation and tendon healing was investigated in 32 New Zealand adult rabbits. The long flexor tendons of the digits of each hind paw were completely divided and repaired with a modified Kessler technique. The rabbits were randomly divided into 4 experimental groups according to the type of repair used: sheath excision, sheath excision and local HAF application, sheath repair, and sheath repair and local HAF application. The extent of adhesions and the healing status of the tendons were macroscopically and histologically evaluated at 12 weeks. Tensile strength of the repaired tendons was measured biomechanically at 20 weeks. The least adhesion and the best healing were observed in tendons treated with sheath repair and HAF application. Tendons treated with HAF had significantly higher tensile load values. Topical application of HAF immediately after tenorrhaphy is significantly effective in preventing peritendinous adhesion formation without impairment of tendon healing in this rabbit model.