The calcaneal tendon (CT) is the most commonly injured tendon in the human body. Moreover, with advancing age, the amount of damage increases further. Resistance training (RT) could be used to minimize such damages. The aim of the present study was to obtain the identification, detailed protein cataloging and biochemical characterization based on the effects of the aging process and the RT in CT of rats. The analysis by liquid chromatography tandem-mass spectrometry showed 142 distinct proteins, however, only 29 proteins met the inclusion criteria and were analyzed. Aging causes a reduction in the abundance of seven proteins related to extracellular matrix organization, biological regulation and cellular processes. However, RT promoted the positive regulation of proteins important for the maintenance of healthy tendons: seven proteins in young trained and two in older trained group. This study contributes to a better understanding of molecular aspects of the tendon. The down regulation of proteins linked to mechanical function and extracellular matrix remodeling of the tendon during aging can contribute to the increase of injury and weakness in the tendon. Nevertheless, RT proved to be a tool to prevent these adverse effects during aging by increasing proteins involved in the functionality of the tendon.