BACKGROUND: Intestinal transplantation (ITx) has the highest rate of rejection among solid organ grafts. We aimed to study the pathophysiology of rejection after ITx but lacked a tool for assessing cellular responses within the graft. Therefore, we developed a novel mixed lymphocyte reaction (MLR) assay to investigate immune responses in the graft. METHODS: Intestinal samples were collected, decontaminated, and processed into single-cell suspensions from 9 swine and 2 patients that underwent ITx. Debris was removed using gradient centrifugation. The cells were plated with corresponding stimulator cells and incubated for 6 d before data acquisition and analysis. RESULTS: Tolerant animals showed no anti-donor or anti-recipient responses in their graft mucosa but maintained strong anti-third-party responses, even after weaning immunosuppression. An animal with graft-versus-host disease displayed robust anti-recipient and anti-third-party responses but no anti-donor response. The animals with graft rejection maintained anti-donor responses at all timepoints. Finally, some tolerant animals developed "split tolerance," with anti-donor responses in the peripheral blood but donor-specific hyporesponsiveness in the mucosal MLR, which regulatory T cells depletion suggested was attributable to local regulatory tolerance. When applied to human sample, this mucosal MLR reliably demonstrated self-tolerance with normal anti-third-party responsiveness. CONCLUSIONS: The novel mucosal MLR assay presented herein ± CD25 depletion serves as a useful adjunct for assessing immune responses within the intestinal graft mucosa. This could help elucidate immune responses after ITx in future studies, including our own, and could represent a promising tool for studying ITx tolerance development, guiding immunosuppression strategies, and advancing personalized transplant medicine.