Background: The major limitation to the Ross operation is a progressive autograft dilation, possibly leading to reoperations. A murine model was created to evaluate pulmonary artery graft (PAG) adaptation to pressure overload. Methods: Lewis rats (n = 17) underwent heterotopic surgical implantation of a PAG, harvested from syngeneic animals (n = 17). A group of sham animals (n = 7) was used as a control. Seriated ultrasound studies of the PAG were performed. Animals were sacrificed at 1 week (n = 5) or 2 months (n = 15) and the PAG underwent mechanical and histopathological analyses. Results: Echography showed an initial increase in diameter (p < 0.001) and a decrease in peak systolic velocity (PSV). Subsequently, despite no change in diameter, an increase in PSV was observed (p < 0.01). After 1 week, the stiffness of the PAG and the aorta were similar, while at 2 months, the PAG appeared more rigid (p < 0.05). PAG’s histological analysis at 2 months revealed intimal hyperplasia development. The tunica media showed focal thinning of the elastic lamellae and normally distributed smooth muscle cells. Conclusions: We demonstrated a stiffening of the PAG wall after its implantation in systemic position; the development of intimal hyperplasia and the thinning of the elastic lamellae could be the possible underlying mechanism.