Background and aims The alpha-galactosidase (αGal) epitope and the associated anti-Gal antibodies, along with the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) and its corresponding anti-Neu5Gc antibodies, represent critical obstacles in the field of xenotransplantation. We present an evaluation of serological and histopathological data from patients who experienced immunological rejection after receiving decellularized xenografts. This study aims to analyze the long-term immune responses that occur following the implantation of these grafts, providing insights into the mechanisms driving rejection and their potential impact on transplant outcomes. Methods Decellularized xenografts, such as bovine pericardium and porcine pulmonary artery treated with a novel patented processing technique, were utilized in cardiovascular surgeries over the course of a decade. Serum samples from patients following xenotransplantation were examined for α-Gal and Neu5Gc titers after various follow-up periods. When explanted during later surgeries, the xenograft materials were assessed histologically using hematoxylin and eosin (H&E), von Kossa, and Movat's pentachrome staining. Immunohistochemical staining for von Willebrand Factor (vWF), alpha-smooth muscle actin (αSMA), and B lymphocyte antigen CD19 was performed to evaluate endothelialization, calcification, fibrosis, and immune rejection. No healthy volunteers were enrolled as study participants. However, for the purpose of baseline serological comparison, serum samples from one healthy adult blood donor (33/M) were used as the standard. This donor had no history of xenograft implantation or known immune disorders. Results and conclusion Histological evaluation showed human cell infiltration within the decellularized xenograft scaffold, with evidence of reendothelialization on the luminal surface. The neointimal layer had variable thickness, with aligned collagen fibers. Early remodeling was indicated by minimal fibroblastic invasion and the presence of microcapillaries. von Kossa staining revealed insignificant calcium deposits, and immunohistochemistry showed minimal signs of immune rejection. Serological analysis of seven patients revealed varying responses to α-Gal and Neu5Gc. The patients had a mean age of 16.57 years (ranging from three to 52 years) and a mean follow-up period of 6.14 years (ranging from four to nine years) at the time of blood sampling. The study included four patients with bovine pericardium grafts, one with a bovine jugular vein graft, and two with porcine pulmonary artery grafts. Anti-αGal antibody levels were consistent across all patients, while two patients showed no anti-Neu5Gc antibodies. The remaining five patients exhibited titers with a high standard deviation. This in vitro evaluation indicates that decellularized xenografts processed using the patented technology provoke minimal tissue and immune responses, making them safe and durable options for cardiovascular remodeling surgeries.