Injuries to skeletal ligaments are prevalent and debilitating. After an injury, ligaments fail to repair and a scar tissue is formed that lacks the mechanical and functional properties of the normal intact ligament. Previously, we have shown that application of the recombinant human amelogenin protein (rHAM+) alone, induced full mechanical and histological healing of the knee medial collateral ligament (MCL) in-vivo after complete transection. The MCL transection model is a common proof of concept model for ligament healing. The aim of this study was to evaluate whether rHAM+ can induce regeneration of the ankle calcaneo-fibular ligament (CFL) tear, which is a major unmet clinical need. Rat`s right ankle CFL was isolated, transected and treated with 0.5µg/µl rHAM+ dissolved in Propylene Glycol Alginate (PGA) comparing to a control group treated with PGA alone. 3 months after treatment, the mechanical properties were evaluated. Histological analysis and SEM were also conducted to demonstrate the microscopic structure of the treated ligament compared to the control group. Our results show significant improvement in the mechanical properties of the CFL treated with rHAM+ similar to the mechanical strength of normal untouched ligaments and much stronger than the control, treated with PGA carrier alone. In the experimental- rHAM+ treated group, the collagen fibers were aligned along with the direction of force, compared to the control group, which resembled a scar tissue. These results indicate that a single application of rHAM+ after CFL transection causes complete healing of the ligament. To date, no registered therapeutic agent enables regeneration of injured ligaments. The simplicity and high potency of this therapeutic option surpasses all the modalities currently used.